US10913798B2 - Modulators of ROR1-ROR2 binding - Google Patents

Modulators of ROR1-ROR2 binding Download PDF

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US10913798B2
US10913798B2 US15/568,737 US201615568737A US10913798B2 US 10913798 B2 US10913798 B2 US 10913798B2 US 201615568737 A US201615568737 A US 201615568737A US 10913798 B2 US10913798 B2 US 10913798B2
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Thomas J. Kipps
Jian Yu
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2857—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, orphan receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures

Definitions

  • ROR1 and ROR2 are evolutionarily conserved type I proteins[1-8]. Studies on the developmental expression patterns of these proteins in Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis , and Mus musculus have shown striking conservation[9]. ROR1 and ROR2 are expressed at highest levels during the early stages of embryogenesis, being represented in most of the major systems in tissues derived from all three germ layers, but most prominently the neural crest, whereas expression of ROR1 appears more restricted to the neural mesenchyme[10,11]. Complete knockout of either ROR1 or ROR2, however, results in pervasive developmental abnormalities involving also the heart, lungs, urogenital tracts, and other organs, suggesting that each potentially contributes broadly to organogenesis [12,13].
  • ROR1 Although low-levels of ROR2 can be found on some adult tissues, post-partum expression of ROR1 is not apparent, except on a small subset of precursor B-cells called hematogones [14]. However, ROR1 can be found on the leukemia cells of patients with chronic lymphocytic leukemia (CLL) [15-17], and ROR1 or ROR2 is expressed by neoplastic cells of a variety of different cancers [18,19]. Cancer-cell expression of ROR1 or ROR2 has been associated with enhanced cancer-cell migration, the epithelial-mesenchymal transition (EMT), increased associated-risk for relapsed disease and metastasis, and unfavorable prognosis [20,21]. More recently, ROR1 was identified on ovarian cancer-stem-cells, which have enhanced capacity for migration/spheroid formation in vitro and engraftment/metastasis in vivo [22].
  • EMT epithelial-mesenchymal
  • ROR1 and ROR2 each may function as a receptor for Wnt5a [15,23], which may induce non-canonical Wnt signaling, potentially leading to enhanced tumor-cell growth, directional migration, and/or tissue-cell polarity during organogenesis [18,24-26].
  • ROR2 also can repress transcription of Wnt-target genes and modulate Wnt signaling by sequestering canonical Wnt ligands, thereby serving as a tumor suppressor in different cell contexts [27].
  • the activity of ROR1 or ROR2 is thought to be influenced by associated proteins [28,29], ROR1 and ROR2 are considered to function independently from one another.
  • a method of identifying an inhibitor of ROR1-ROR2 binding includes (i) combining a test agent with a ROR1 protein and a ROR2 protein in a reaction vessel. (ii) A decrease in binding of the ROR1 protein to the ROR2 protein relative to a standard control is detected and thereby an inhibitor of ROR1-ROR2 binding is identified.
  • an antibody identified by the method provided herein including embodiments thereof is provided.
  • a method of inhibiting a ROR1-ROR2 interaction includes (i) contacting a compound identified by the method provided herein including embodiments thereof with a ROR1-ROR2 complex, wherein the complex includes a ROR1 protein and a ROR2 protein. (ii) The compound is allowed to inhibit the interaction between the ROR1 protein and the ROR2 protein, thereby inhibiting a ROR1-ROR2 interaction.
  • a method of treating cancer in a subject in need thereof includes administering to a subject a therapeutically effective amount of an antibody, small molecule, peptide or nucleic acid identified by a method provided herein including embodiments thereof.
  • a method of identifying an inhibitor of a protein complex including ROR1 and ROR2 proteins includes (i) combining a test agent in a reaction vessel including ROR1 and ROR2; and (ii) detecting a disruption of a protein complex including ROR1 and ROR2 relative to a standard control, thereby identifying an inhibitor of the protein complex including ROR1 and ROR2 proteins.
  • the detecting includes detecting a decreased level of ROR1 and/or ROR2 in the protein complex in the presence of the test agent, wherein a decreased level of ROR1 and/or ROR2 in the complex relative to a standard control indicates the test agent is an inhibitor of a protein complex including ROR1 and ROR2 proteins.
  • the detecting includes detecting an increased level of unbound ROR1 and/or unbound ROR2 in the protein complex in the presence of the test agent, wherein an increased level of unbound ROR1 and/or ROR2 relative to a standard control indicates the test agent is an inhibitor of a protein complex including ROR1 and ROR2 proteins.
  • the ROR1 and/or the ROR2 proteins include a detectable moiety.
  • the detecting includes detecting a level of a guanine exchange factor (GEF) in the presence of the test agent, wherein a decreased level of the GEF relative to a standard control indicates the test agent is an inhibitor of a protein complex having ROR1 and ROR2 proteins.
  • GEF guanine exchange factor
  • the GEF is RhoA, Rac1, AHRGEF1, AHRGEF2 or AHRGEF6.
  • the GEF includes a detectable moiety.
  • the combining occurs in the presence of a Wnt5a protein.
  • the reaction vessel is a column including a solid support.
  • the protein complex comprising ROR1 and ROR2 is present in the absence of a cell. In embodiments, the ROR1 and the ROR2 are present on the surface of a cell.
  • the cell is a tumor cell. In embodiments, the tumor cell is a chronic lymphocytic leukemia (CLL) cell. In embodiments, the CLL cell is a MEC1 cell. In embodiments, the cell forms part of an in vitro cell culture. In embodiments, the cell forms part of an organism. In embodiments, the organism is a mammal. In embodiments, the organism is a mouse.
  • CLL chronic lymphocytic leukemia
  • the test agent is an antibody, a small molecule, a peptide, a protein or a nucleic acid.
  • the test agent is an antibody.
  • the antibody is an anti-ROR1 antibody.
  • the antibody is an anti-ROR2 antibody.
  • the antibody binds to an ROR1 protein or an ROR2 protein.
  • the antibody binds to a KNG domain.
  • the antibody is a humanized antibody.
  • the antibody is a chimeric antibody.
  • the antibody is a scFv.
  • FIGS. 1A-1J Wnt5a enhances CLL proliferation and migration through ROR1-dependent activation of Rac1 and RhoA.
  • FIG. 1B The bars indicate the mean proportions of CLL cells with diminished CFSE fluorescence from each of 6 different patients for each culture condition indicated at the bottom.
  • FIG. 1D Activated Rac1 and RhoA were measured in CLL cells, which were cultured in serum-free media (serum starved) and then treated with Wnt5a for the times indicated above each lane. Whole-cell lysates also were examined via immunoblot analysis for total Rac1 or RhoA.
  • FIG. 1E Wnt5a-induced activation of Rac1 (top panels) or RhoA (bottom panels) in CLL cells treated with Ctrl-IgG or UC-961, as indicated on top of each set of blots.
  • FIG. 1F Activated Rac1 in serum-starved CLL cells treated with or without Wnt5a and/or CD154 for 30 min.
  • FIG. 1G Activated RhoA in serum-starved CLL cells treated with or without Wnt5a and/or CXCL12 for 30 min.
  • FIGS. 1A and 11 The results of assays on CFSE-labeled CLL cells of one representative sample are presented as in panel FIGS. 1A and 11 .
  • FIGS. 2A-2G ROR1 couples with ROR2.
  • FIG. 2A Immunoblot analysis for ROR1 or ROR2 in lysates of ZAP70 Neg /mu-IgVH (CLL-1-4) or ZAP70 + /um-IgVH (CLL-5-8) CLL cells, or peripheral blood mononuclear cells (PBMC) of healthy subjects. Purified recombinant extracellular ROR1 or ROR2 (ROR1-ex or ROR2-ex) were loaded onto separate lanes as specificity controls.
  • FIG. 2B Detection of ROR1 or ROR2 on CD5 + CD19 + CLL cells by flow cytometry.
  • FIG. 1A Immunoblot analysis for ROR1 or ROR2 in lysates of ZAP70 Neg /mu-IgVH (CLL-1-4) or ZAP70 + /um-IgVH (CLL-5-8) CLL cells, or peripheral blood mononuclear cells (PBMC) of healthy
  • FIG. 2C PBMC of healthy donors were stained with anti-ROR2-Alexa-488, anti-CD19-PE, and anti-CD5-APC mAbs and analyzed by flow cytometry.
  • the gating strategy is indicated in the center contour plot for each subgroup specified on the top of each histogram depicting the fluorescence of cells incubated with the anti-ROR2 mAb (shaded) versus an Alexa-488-conjugated Ctrl-IgG (open histograms).
  • the ⁇ MFI for each of the CD19 + cell-subsets are indicated in the top right.
  • FIG. 2E Immunoblot analysis of anti-ROR1 or anti-ROR2 ip from lysates of freshly-isolated CLL cells detecting the association of ROR1 with ROR2.
  • FIG. 2F Immunoblot analysis of anti-ROR1 ip from lysates of CLL cells cultured in serum-free media and then treated without ( ⁇ ) or with (+) Wnt5a.
  • FIGS. 3A-3D UC-961 inhibits ROR1 signaling.
  • FIG. 3A Co-localization (arrow) of ROR1 and ROR2 detected by confocal microscopy in freshly isolated CLL cells+/ ⁇ Ctrl-IgG or UC-961, as indicated on the right margin of each row (Scale bar: 2 ⁇ m).
  • FIG. 3B Confocal microscopy of serum-starved CLL cells stained for ROR1 and ROR2 after treatment with or without Wnt5a and Ctrl-IgG or UC-961, presented as in FIG. 3A .
  • FIG. 3A Co-localization (arrow) of ROR1 and ROR2 detected by confocal microscopy in freshly isolated CLL cells+/ ⁇ Ctrl-IgG or UC-961, as indicated on the right margin of each row (Scale bar: 2 ⁇ m).
  • FIG. 3B Confocal microscopy of serum-starved CLL cells stained for ROR
  • FIG. 3D Activated RhoA or Rac1 were measured by Rho-family protein activity pull-down assays on lysates of CLL cells transfected with Ctrl-siRNA or siRNA specific for ROR1 or ROR2 and cultured with or without Wnt5a. Whole-cell lysates also were examined via immunoblot for total RhoA or Rac1 (bottom row). The numbers below each lane are the densities of activated to total GTPase normalized for untreated samples, as in FIG. 1D .
  • FIGS. 4A-4F UC-961 inhibits Wnt5a-induced recruitment of GEFs and activation of RhoA and Rac1.
  • FIG. 4A Co-localization (arrow) of ARHGEF1 with ROR1 and ROR2 in CLL cells cultured without ( ⁇ ) or with (+) Wnt5a, as indicated on the left margin or each row (Scale bar: 5 ⁇ m).
  • FIG. 4B Co-localization of ARHGEF2 with ROR1-ROR2 in CLL cells cultured without ( ⁇ ) or with (+) Wnt5a, as in FIG. 4A .
  • FIG. 4A Co-localization of ARHGEF2 with ROR1-ROR2 in CLL cells cultured without ( ⁇ ) or with (+) Wnt5a, as in FIG. 4A .
  • FIG. 4C Co-localization of ARHGEF6 with ROR1-ROR2 in CLL cells cultured without ( ⁇ ) or with (+) Wnt5a, as in FIG. 4A .
  • FIG. 4D In vitro exchange over time (in minutes) of RhoA (top two rows of graphs) or Rac1 (bottom two row of graphs) of ip from lysates of CLL cells cultured with or without Wnt5a, using mAbs specific for ARHGEF1, ARHGEF2, or ARHGEF6, as indicated in the bottom right of each graph. The line depicts GTPase-activation observed using buffer alone.
  • FIG. 4E In vitro exchange over time (in minutes) of RhoA (top two rows of graphs) or Rac1 (bottom two row of graphs) of ip from lysates of CLL cells cultured with or without Wnt5a, using mAbs specific for ARHGEF1, ARHGEF2, or ARHGEF6, as indicated in the bottom right of each graph. The line
  • FIG. 4F Activation RhoA or Rac1 following treatment without ( ⁇ ) or with (+) Wnt5a of CLL cells transfected with Ctrl-siRNA or siRNA specific for ARHGEF1, ARHGEF2, or ARHGEF6.
  • Whole-cell lysates also were examined via immunoblot analysis for total RhoA or Rac1. The numbers beneath each lane is the ratio of band densities for activated versus total GTPase normalized for untreated samples.
  • FIGS. 5A-5I Mapping the functional domains of ROR1.
  • FIG. 5A Schematic representation of the structure of ROR1 and truncated forms of ROR1 was shown. Interaction of ROR1 with ROR2 was confirmed by immunoblot analysis of anti-ROR1 ip using lysates from MEC1 (Ctrl.), MEC1-ROR1 (ROR1), or MEC1 cells transfected with truncated forms of ROR1.
  • FIG. 5B Co-localization (e.g., arrow) of ARHGEF1, ARHGEF2 or ARHGEF6 with ROR1 and ROR2 in MEC1-ROR1 cells (Scale bar: 2 ⁇ m).
  • FIG. 5A Schematic representation of the structure of ROR1 and truncated forms of ROR1 was shown. Interaction of ROR1 with ROR2 was confirmed by immunoblot analysis of anti-ROR1 ip using lysates from MEC1 (Ctrl.), M
  • FIG. 5C Activated Rac1 or RhoA were measured in MEC1 (Ctrl.), MEC1-ROR1 (ROR1), and MEC1 cells expressing truncated forms of ROR1. Whole-cell lysates also were examined via immunoblot for total Rac1 or RhoA. The numbers beneath each lane are the ratios of band densities for activated versus total GTPase normalized for MEC1 samples.
  • FIG. 5D Inhibition of Rac1- or RhoA-activation by UC-961 and anti-Wnt5a mAbs in MEC1 or MEC1-ROR1 cells. The numbers beneath each lane are the ratios of band densities normalized for MEC1, as in FIG. 5C .
  • FIG. 5E The mean numbers of MEC1 (circles), MEC1-ROR1 (squares), or MEC1 cells expressing each of the truncated forms of ROR1 (as indicated in the legend) in triplicate wells at the days indicated below the graph.
  • FIG. 5F The mean numbers of MEC1-ROR1 cells cultured with Ctrl-IgG (squares), UC-961 (triangles) or anti-Wnt5a (inverted triangles) in triplicate wells at the times indicated.
  • FIG. 5E The mean numbers of MEC1 (circles), MEC1-ROR1 (squares), or MEC1 cells expressing each of the truncated forms of ROR1 (as indicated in the legend) in triplicate wells at the days indicated below the graph.
  • FIG. 5F The mean numbers of MEC1-ROR1 cells cultured with Ctrl-IgG (squares), UC-961 (triangles) or anti-Wnt5a (in
  • FIG. 5G The bars indicate the mean proportions of MEC1, MEC1-ROR1, or MEC1 cells transfected with each of the truncated forms of ROR1 (as indicated in the legend) migrating with (+) or without ( ⁇ ) CCL21, as indicated at the bottom.
  • FIG. 5H Bars depict the mean proportions of MEC1-ROR1 cells migrating with (+) or without ( ⁇ ) CCL21 in the presence of Ctrl-IgG, UC-961 or anti-Wnt5a. Data in FIGS. 5E-5H are shown as mean ⁇ SEM; *P ⁇ 0.05; **P ⁇ 0.01.
  • FIG. 5I Cartoon diagram modeling molecular mechanisms by which Wnt5a enhances proliferation and migration by inducing formation of a ROR1-ROR2 heterodimer, which recruits GEFs that activate Rac1 and RhoA.
  • FIGS. 6A-6E UC-961 inhibits engraftment of MEC1-ROR1.
  • FIG. 6A Representative spleens of Rag2 ⁇ / ⁇ ⁇ c ⁇ / ⁇ mice 3 weeks after receiving an intravenous infusion of 1 ⁇ 10 6 MEC1 or MEC1-ROR1 cells. The spleen of an age-matched, non-engrafted Rag2 ⁇ / ⁇ ⁇ c ⁇ / ⁇ mouse (Nor) is shown for comparison.
  • FIG. 6A Representative spleens of Rag2 ⁇ / ⁇ ⁇ c ⁇ / ⁇ mice 3 weeks after receiving an intravenous infusion of 1 ⁇ 10 6 MEC1 or MEC1-ROR1 cells. The spleen of an age-matched, non-engrafted Rag2 ⁇ / ⁇ ⁇ c ⁇ / ⁇ mouse (Nor) is shown for comparison.
  • MEC1 or MEC1-ROR1 cells were collected from the marrow of the femur or tibia or from the spleens of mice engrafted 3-weeks earlier with MEC1 or MEC1-ROR1 cells, as indicated on the right margin.
  • the fluorescence of cells stained with 4A5-Alexa-647 (ordinate) and anti-CD19-PE (abscissa) are shown in the contour plots. The percentages at the top right of each contour plot indicate the proportions of cells with fluorescence above the threshold indicated by the dotted line.
  • FIG. 6C Representative spleens of Rag2 ⁇ / ⁇ ⁇ c ⁇ / ⁇ mice 3 weeks after receiving an intravenous infusion of 1 ⁇ 10 6 MEC1-ROR1 cells and then treatment with either Ctrl-IgG or UC-961.
  • the spleen of an age-match non-engrafted Rag2 ⁇ / ⁇ ⁇ c ⁇ / ⁇ mouse (Nor) is shown for comparison.
  • FIG. 6D MEC1-ROR1 cells were collected from the marrow of the femur and tibia or the spleens of mice engrafted 3-weeks earlier with MEC1-ROR1 and then treated with either Ctrl-IgG or UC-961, as indicated on the right.
  • FIGS. 8A-8E ROR2 expression in CLL cells.
  • FIG. 8A Mass spectrum showing unique peptide of ROR2 (SEQ ID NO:24) identified by 2D-nanoLC-MS/MS from the protein lysates prepared from CLL cells following ip with anti-ROR1 mAb versus Ctrl-IgG.
  • FIG. 8B Total RNA was extracted from each of 3 CLL patient samples. ROR2 cDNA was amplified by RT-PCR and confirmed by DNA sequencing. Total RNA was used as PCR template to ensure there was no genomic DNA contamination.
  • FIG. 8D ROR1-ex and ROR2-ex proteins were purified and analyzed by SDS-PAGE and Coomassie staining.
  • FIG. 8E Chinese hamster ovary (CHO) cells were transfected with pcDNA3.1 (Vector, left panel) or pcDNA3.1-encoding ROR2 (ROR2, right panel) and then stained with a Alexa-488-labeled isotype control mAb (open histograms) or an anti-ROR2-Alexa-488 mAb for analysis by flow cytometry.
  • FIG. 9 Negative control for co-localization analysis by confocal microscopy.
  • CLL cells were stained with anti-CD5-Alexa-647 and anti-CD19-Alexa-488; anti-ROR1-Alexa-647 and anti-CD19-Alexa-488; or anti-CD5-Alexa-647 and anti-ROR2-Alexa-488 for evaluation by confocal microscopy (Scale bar: 2 ⁇ m). There is no co-localization between CD5 and CD19, ROR1 and CD19, or CD5 and ROR2.
  • FIGS. 10A-10B Silencing of ROR1 and ROR2 in CLL.
  • FIG. 10A CLL cells were transfected with non-specific control siRNA (Ctrl-siRNA), or siRNA specific for ROR1 (ROR1-siRNA) or ROR2 (ROR2-siRNA), as indicated at the top of each lane. The cells were examined 48 h later for ROR1 or ROR2 via immunoblot analysis. The numbers beneath each lane is the ratio of band densities for ROR1 or ROR2 versus ⁇ -actin normalized with respect to Ctrl-siRNA treated samples.
  • FIG. 10A CLL cells were transfected with non-specific control siRNA (Ctrl-siRNA), or siRNA specific for ROR1 (ROR1-siRNA) or ROR2 (ROR2-siRNA), as indicated at the top of each lane. The cells were examined 48 h later for ROR1 or ROR2 via immunoblot analysis. The numbers beneath each
  • 10B Flow cytometry of CLL cells transfected with Ctrl-siRNA (black histogram) or either ROR1-siRNA or ROR2-siRNA (grey histograms) and then stained with anti-CD5-Alexa-647 (left panel) or anti-ROR2-Alexa-488 (right panel).
  • the open histograms are representative of the fluorescence of control or transfected CLL cells stained with an Alexa-488-labeled isotype Ctrl-IgG (Isotype).
  • FIGS. 11A-11B ROR1 interacts with ARHGEF1, ARHGEF2, and ARHGEF6.
  • FIG. 11A Mass spectra showing unique peptides of ARHGEF1, ARHGEF2, or ARHGEF6 identified by 2D-nanoLC-MS/MS from the protein lysates prepared from CLL cells following ip with anti-ROR1 mAb versus Ctrl-IgG. Sequence legend: KQLLFPAEEDNGAGPPRD (SEQ ID NO:5); KAAAQGPEGDIQEQELQSEELGLRA (SEQ ID NO:6); WQIWELLSTDNPDALPE (SEQ ID NO:7).
  • FIG. 11A Mass spectra showing unique peptides of ARHGEF1, ARHGEF2, or ARHGEF6 identified by 2D-nanoLC-MS/MS from the protein lysates prepared from CLL cells following ip with anti-ROR1 mAb versus Ctrl-IgG. Sequence legend: KQLL
  • FIG. 11B Immunoblot analysis of ip generated using lysates of freshly isolated CLL cells with a mAb specific for ROR1, ARHGEF1, ARHGEF2, or ARHGEF6, as indicated above each lane. The immunoblots were probed with antibodies specific for ROR1, ARHGEF1, ARHGEF2, or ARHGEF6, as indicated on the left margin of each subpanel.
  • FIGS. 12A-12D UC-961 inhibits co-localization of ARHGEF1, ARHGEF2, or ARHGEF6 with ROR1 or ROR2.
  • FIG. 12A Co-localization (e.g., arrow) of ROR1 with ARHGEF1, ARHGEF2, or ARHGEF6 detected by confocal microscopy in serum-starved CLL cells+/ ⁇ Wnt5a (as indicated on the left margin) and Ctrl-IgG or UC-961 (Scale bar: 5 ⁇ m).
  • FIG. 12B In vitro exchange over time (in minutes) of Rac1 (left panel) or RhoA (right panel) of ip from lysates of CLL cells cultured with or without Wnt5a, using mAbs specific for ARHGEF1 (left subpanel) or ARHGEF6 (right subpanel), as indicated in the bottom right of each graph.
  • the line depicts GTPase-activation using buffer alone.
  • FIG. 12C CLL cells were transiently transfected with non-specific Ctrl-siRNA or siRNA specific for ARHGEF1 (ARHGEF1-siRNA), ARHGEF2 (ARHGEF2-siRNA), or ARHGEF6 (ARHGEF6-siRNA), as indicated at the top of each lane.
  • FIG. 12D Activation of Rac1 or RhoA in CLL cells transfected with Ctrl-siRNA, ARHGEF1-siRNA, or ARHGEF6-siRNA following treatment without ( ⁇ ) or with (+) Wnt5a, as indicated at the top of the lanes.
  • Whole-cell lysates also were examined via immunoblot analysis for total Rac1 or RhoA.
  • the numbers beneath each lane is the ratio of band densities for activated versus total GTPase normalized for untreated, Ctrl-siRNA-transfected samples.
  • FIGS. 13A-13B Expression of ROR1, ROR2, and Wnt5a in parental MEC1 cells and MEC1 cells transfected with each of the various ROR1 constructs.
  • FIG. 13A Fluorescence of MEC1 cells, or MEC1 cells transfected with vectors encoding ROR1 or truncated forms of ROR1, stained with a fluorochrome-labeled isotype control mAb (open histogram) or 4A5-Alexa-647 (top row) or anti-ROR2-Alexa-488 (bottom row) (shaded histograms).
  • FIG. 13B Expression of Wnt5a was determined by immunoblot analysis of lysates from primary CLL cells or MEC1 cells. Recombinant Wnt5a (100 ng) was used as a positive control and ⁇ -actin served as a loading control for cell lysates.
  • FIGS. 14A-14C Confocal microscopy of MEC1 cells made to express ROR1 or various truncated forms of ROR1.
  • FIG. 14A Co-localization (e.g., arrow) of ROR1 and ROR2 in MEC1 cells transfected with each of the various ROR1 constructs, as indicated on the right of each row of figures (Scale bar: 2 ⁇ m).
  • FIG. 14B Co-localization (e.g., arrow) of ROR1 and ARHGEF1, ARHGEF2, or ARHGEF6 in MEC1 cells transfected with each of the various ROR1 constructs, as indicated on the right of each row of figures (Scale bar: 2 ⁇ m).
  • FIG. 14A Co-localization (e.g., arrow) of ROR1 and ARHGEF1, ARHGEF2, or ARHGEF6 in MEC1 cells transfected with each of the various ROR1 constructs, as indicated on the right of each row of figures (Scale bar: 2
  • Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof.
  • polynucleotide refers to a linear sequence of nucleotides.
  • nucleotide typically refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof.
  • nucleic acid as used herein also refers nucleic acids that have the same basic chemical structure as a naturally occurring nucleic acid. Such analogues have modified sugars and/or modified ring substituents, but retain the same basic chemical structure as the naturally occurring nucleic acid.
  • a nucleic acid mimetic refers to chemical compounds that have a structure that is different the general chemical structure of a nucleic acid, but that functions in a manner similar to a naturally occurring nucleic acid.
  • Examples of such analogues include, without limitation, phosphorothiolates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).
  • Synthetic mRNA refers to any mRNA derived through non-natural means such as standard oligonucleotide synthesis techniques or cloning techniques. Such mRNA may also include non-proteinogenic derivatives of naturally occurring nucleotides. Additionally, “synthetic mRNA” herein also includes mRNA that has been expressed through recombinant techniques or exogenously, using any expression vehicle, including but not limited to prokaryotic cells, eukaryotic cell lines, and viral methods. “Synthetic mRNA” includes such mRNA that has been purified or otherwise obtained from an expression vehicle or system.
  • complementarity refers to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide.
  • sequence A-G-T is complementary to the sequence T-C-A.
  • Complementarity may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing.
  • Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding 15 sequence if it is positioned so as to facilitate translation.
  • “operably linked” means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase.
  • polypeptide refers to a polymer of amino acid residues, wherein the polymer may be conjugated to a moiety that does not consist of amino acids.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • the terms apply to macrocyclic peptides, peptides that have been modified with non-peptide functionality, peptidomimetics, polyamides, and macrolactams.
  • a “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.
  • peptidyl and “peptidyl moiety” means a monovalent peptide.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • the terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region, e.g., of the entire polypeptide sequences of the invention or individual domains of the polypeptides of the invention), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • sequences are then said to be “substantially identical.”
  • This definition also refers to the complement of a test sequence.
  • the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol.
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • amino acid or nucleotide base “position” is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to an N-terminus (or 5′-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion.
  • numbered with reference to or “corresponding to,” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to a reference sequence.
  • the comparison to the reference sequence is a sequence alignment between the given amino acid or polynucleotide sequence and the reference sequence.
  • recombinant when used with reference to, for example, a cell, nucleic acid, or protein, indicates that the cell, nucleic acid, or protein, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express genes otherwise modified from those found in the native form of a cell (e.g. genes encoding a mutation in a native or non-native transporter protein, such as a transporter motif sequence as described herein).
  • a recombinant protein may be a protein that is expressed by a cell or organism that has been modified by the introduction of a heterologous nucleic acid (e.g. encoding the recombinant protein).
  • the word “expression” or “expressed” as used herein in reference to a DNA nucleic acid sequence means the transcriptional and/or translational product of that sequence.
  • the level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88).
  • gene means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
  • the leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene.
  • a “protein gene product” is a protein expressed from a particular gene.
  • isolated refers to a nucleic acid, polynucleotide, polypeptide, protein, or other component that is partially or completely separated from components with which it is normally associated (other proteins, nucleic acids, cells, etc.).
  • an isolated polypeptide or protein is a recombinant polypeptide or protein.
  • a “cell culture” is an in vitro population of cells residing outside of an organism.
  • the cell culture can be established from primary cells isolated from a cell bank or animal, or secondary cells that are derived from one of these sources and immortalized for long-term in vitro cultures.
  • a cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring.
  • Cells may include prokaryotic and eukaryotic cells.
  • Prokaryotic cells include but are not limited to bacteria.
  • Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells.
  • transfection can be used interchangeably and are defined as a process of introducing a nucleic acid molecule and/or a protein to a cell.
  • Nucleic acids may be introduced to a cell using non-viral or viral-based methods.
  • the nucleic acid molecule can be a sequence encoding complete proteins or functional portions thereof.
  • a nucleic acid vector comprising the elements necessary for protein expression (e.g., a promoter, transcription start site, etc.).
  • Non-viral methods of transfection include any appropriate method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell.
  • Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation.
  • any useful viral vector can be used in the methods described herein.
  • examples of viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors.
  • the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well known in the art.
  • the terms “transfection” or “transduction” also refer to introducing proteins into a cell from the external environment.
  • transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods 4:119-20.
  • plasmid refers to a nucleic acid molecule that encodes for genes and/or regulatory elements necessary for the expression of genes. Expression of a gene from a plasmid can occur in cis or in trans. If a gene is expressed in cis, gene and regulatory elements are encoded by the same plasmid. Expression in trans refers to the instance where the gene and the regulatory elements are encoded by separate plasmids.
  • episomal plasmids are nucleic acid molecules that are not part of the chromosomal DNA and replicate independently thereof.
  • exogenous refers to a molecule or substance (e.g., nucleic acid or protein) that originates from outside a given cell or organism.
  • endogenous refers to a molecule or substance that is native to, or originates within, a given cell or organism.
  • a “vector” is a nucleic acid that is capable of transporting another nucleic acid into a cell.
  • a vector is capable of directing expression of a protein or proteins encoded by one or more genes carried by the vector when it is present in the appropriate environment.
  • telomere binding refers to two molecules forming a complex (e.g., a protein complex comprising a ROR1 and a ROR2 protein, or a protein complex comprising ROR1 and/or a ROR2 protein bound to an antibody) that is relatively stable under physiologic conditions.
  • a ligand e.g., antibody
  • a protein e.g., protein
  • affinity for a ligand to a protein e.g., the binding of a ligand to a protein can be detected and/or quantified using a variety of techniques such as, but not limited to, Western blot, dot blot, surface plasmon resonance method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.), bio-layer interferometry (BLI) technology (e.g., Octet system; Pall ForteBio Menlo Park, Calif.), isothermal titration calorimetry (ITC), or enzyme-linked immunosorbent assays (ELISA).
  • Western blot e.g., dot blot, surface plasmon resonance method
  • BIAcore surface plasmon resonance method
  • ITC isothermal titration calorimetry
  • ELISA enzyme-
  • Immunoassays which can be used to analyze immunospecific binding and cross-reactivity of the ligand include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, RIA, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, and fluorescent immunoassays.
  • competitive and non-competitive assay systems using techniques such as Western blots, RIA, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, and fluorescent immunoassays.
  • a “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins (e.g., antibodies) or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or covalently attaching a fluorophore to an antibody specifically reactive with a target peptide. Any appropriate method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.
  • a “labeled protein or polypeptide” is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the labeled protein or polypeptide may be detected by detecting the presence of the label bound to the labeled protein or polypeptide.
  • methods using high affinity interactions may achieve the same results where one of a pair of binding partners binds to the other, e.g., biotin, streptavidin.
  • a “control” sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample.
  • a test sample can be taken from a test condition, e.g., in the presence of a test compound, and compared to samples from known conditions, e.g., in the absence of the test compound (negative control), or in the presence of a known compound (positive control).
  • a control can also represent an average value gathered from a number of tests or results.
  • controls can be designed for assessment of any number of parameters. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects).
  • Standard controls are also valuable for determining the significance (e.g. statistical significance) of data. For example, if values for a given parameter are widely variant in standard controls, variation in test samples will not be considered as significant.
  • inhibitor refers to a substance capable of detectably decreasing the expression or activity of a given gene or protein.
  • the antagonist can decrease expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more in comparison to a control in the absence of the antagonist or an antagonist can decrease expression or activity by an amount that is within a range defined by any two of the aforementioned percentages in comparison to a control in the absence of the antagonist.
  • expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold lower than the expression or activity in the absence of the antagonist or expression or activity is within a range defined by any two of the aforementioned values lower than the expression or activity in the absence of the antagonist.
  • the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to a protein-inhibitor (e.g., test agent as provided herein) interaction means negatively affecting (e.g., decreasing) the activity or function of the protein (e.g., decreasing the binding of ROR1 to ROR2 or vice versa e.g., in a protein complex comprising ROR1 and ROR2) relative to the activity or function of the protein in the absence of the inhibitor (e.g., test agent).
  • inhibition refers to reduction of a disease or symptoms of disease.
  • inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein.
  • an “inhibitor” is a compound or protein that inhibits ROR1 or ROR2 activity, e.g., by binding, partially or totally blocking, decreasing, preventing, delaying, inactivating, desensitizing, or down-regulating enzymatic activity (e.g., ROR1 or ROR2 catalytic activity).
  • small molecule refers to a compound having a molecule weight of 500 Daltons or less, but greater than 1 Dalton. Although small molecules may be of biological original, small molecules are typically non-physiologic.
  • Antibodies are large, complex molecules (molecular weight of ⁇ 150,000 or about 1320 amino acids) with intricate internal structure.
  • a natural antibody molecule contains two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain.
  • Each light chain and heavy chain in turn consists of two regions: a variable (“V”) region involved in binding the target antigen, and a constant (“C”) region that interacts with other components of the immune system.
  • the light and heavy chain variable regions come together in 3-dimensional space to form a variable region that binds the antigen (for example, a receptor on the surface of a cell).
  • Within each light or heavy chain variable region there are three short segments (averaging 10 amino acids in length) called the complementarity determining regions (“CDRs”).
  • the six CDRs in an antibody variable domain fold up together in 3-dimensional space to form the actual antibody binding site which docks onto the target antigen.
  • the position and length of the CDRs have been precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987.
  • the part of a variable region not contained in the CDRs is called the framework (“FR”), which forms the environment for the CDRs.
  • CDR complementarity determining region
  • CDRL1, CDRL2, and CDRL3 the regions that are primarily responsible for antigen-binding.
  • CDRH1, CDRH2, and CDRH3 the residues forming the six CDRs are residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variable region and 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) in the heavy chain variable region.
  • the residues forming the six CDRs are residues 26-32 (CDRL1), 50-52 (CDRL2) and 91-96 (CDRL3) in the light chain variable region and 26-32 (CDRH1), 53-55 (CDRH2) and 96-101 (CDRH3) in the heavy chain variable region. Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917, herein incorporated by reference. Unless specified, as used herein, the numbering of CDR residues is according to Kabat.
  • antibody is used according to its commonly known meaning in the art.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′ 2 , a dimer of Fab which itself is a light chain joined to V H -C H1 by a disulfide bond.
  • the F(ab)′ 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′ 2 dimer into an Fab′ monomer.
  • the Fab′ monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3 d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).
  • antibody fragments refers to a portion of an antibody.
  • antibody fragments include, but are not limited to, linear antibodies, single-chain antibody molecules, Fv, Fab and F(ab′).sub.2 fragments, and multispecific antibodies formed from antibody fragments.
  • the antibody fragments retain at least part of the heavy and/or light chain variable region.
  • mAb monoclonal antibodies
  • Techniques for the production of single chain antibodies can be adapted to produce antibodies to polypeptides disclosed herein.
  • phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens.
  • a humanized antibody is a genetically engineered antibody in which at least one CDR (or functional fragment thereof) from a mouse antibody (“donor antibody”, which can also be rat, hamster or other non-human species) are grafted onto a human antibody (“acceptor antibody”). In embodiments, more than one mouse CDR is grafted (e.g. all six mouse CDRs are grafted).
  • the sequence of the acceptor antibody can be, for example, a mature human antibody sequence (or fragment thereof), a consensus sequence of a human antibody sequence (or fragment thereof), or a germline region sequence (or fragment thereof).
  • a humanized antibody may be an antibody having one or more CDRs from a donor antibody and a variable region framework (FR).
  • amino acids in the human acceptor sequence may be replaced by the corresponding amino acids from the donor sequence, for example where: (1) the amino acid is in a CDR; (2) the amino acid is in the human framework region (e.g. the amino acid is immediately adjacent to one of the CDR's).
  • the amino acid is in a CDR
  • the amino acid is in the human framework region (e.g. the amino acid is immediately adjacent to one of the CDR's).
  • humanized antibodies often incorporate all six CDRs (e.g. as defined by Kabat, but often also including hypervariable loop H1 as defined by Chothia) from a mouse antibody, they can also be made with fewer mouse CDRs and/or less than the complete mouse CDR sequence (e.g.
  • a functional fragment of a CDR e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al, Journal of Immunology, 164:1432-1441, 2000.
  • a humanized antibody as provided herein may include (i) a light chain comprising at least one CDR (often three CDRs) from a mouse antibody (also referred to herein as a mouse CDR) and a human variable region framework; and (ii) a heavy chain comprising at least one CDR (often three CDRs) from the mouse antibody and a human variable region framework (FR).
  • the light and heavy chain variable region frameworks (FRs) may each be a mature human antibody variable region framework sequence (or fragment thereof), a germline variable region framework sequence (combined with a J region sequence) (or fragment thereof), or a consensus sequence of a human antibody variable region framework sequence (or fragment thereof
  • a chimeric antibody is an antibody in which the variable region of a mouse (or other rodent) antibody is combined with the constant region of a human antibody; their construction by means of genetic engineering is well-known. Such antibodies retain the binding specificity of the mouse antibody, while being about two-thirds human. The proportion of nonhuman sequence present in mouse, chimeric and humanized antibodies suggests that the immunogenicity of chimeric antibodies is intermediate between mouse and humanized antibodies.
  • Other types of genetically engineered antibodies that may have reduced immunogenicity relative to mouse antibodies include human antibodies made using phage display methods (Dower et al., WO91/17271; McCafferty et al., WO92/001047; Winter, WO92/20791; and Winter, FEBS Lett. 23:92, 1998, each of which is incorporated herein by reference) or using transgenic animals (Lonberg et al., WO93/12227; Kucherlapati WO91/10741, each of which is incorporated herein by reference).
  • the epitope of a mAb is the region of its antigen to which the mAb binds.
  • Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1 ⁇ , 5 ⁇ , 10 ⁇ , 20 ⁇ or 100 ⁇ excess of one antibody or an amount of one antibody that is within a range defined by any two of the aforementioned amounts inhibits binding of the other by at least 30% but preferably 50%, 75%, 90% or even 99% or within a range defined by any two of the aforementioned values, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990).
  • two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
  • Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
  • ROR1 protein as referred to herein includes any of the recombinant or naturally-occurring forms of the tyrosine-protein kinase transmembrane receptor (ROR1) also known as neurotrophic tyrosine kinase receptor-related 1 (NTRKR1) or variants or homologs thereof that maintain ROR1 kinase activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity or within a range of activity defined by any two of the aforementioned values compared to ROR1).
  • ROR1 protein includes any of the recombinant or naturally-occurring forms of the tyrosine-protein kinase transmembrane receptor (ROR1) also known as neurotrophic tyrosine kinase receptor-related 1 (NTRKR1) or variants or homologs thereof that maintain ROR1 kinase activity (e.g. within at least 50%, 80%,
  • the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity or within a range of identities defined by any two of the aforementioned values across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion or within a range defined by any two of the aforementioned lengths) compared to a naturally occurring ROR1 protein.
  • the ROR1 protein is substantially identical to the protein identified by the UniProt reference number Q01973 (SEQ ID NO:8) or a variant or homolog having substantial identity thereto.
  • the ROR1 protein is substantially identical to the protein identified by the UniProt reference number Q9Z139 (SEQ ID NO:9) or a variant or homolog having substantial identity thereto.
  • ROR2 protein as referred to herein includes any of the recombinant or naturally-occurring forms of the tyrosine-protein kinase transmembrane receptor (ROR2) also known as neurotrophic tyrosine kinase receptor-related 1 (NTRKR2) or variants or homologs thereof that maintain ROR2 kinase activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity or within a range of activity defined by any two of the aforementioned values compared to ROR2).
  • ROR2 protein includes any of the recombinant or naturally-occurring forms of the tyrosine-protein kinase transmembrane receptor (ROR2) also known as neurotrophic tyrosine kinase receptor-related 1 (NTRKR2) or variants or homologs thereof that maintain ROR2 kinase activity (e.g. within at least 50%, 80%,
  • the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity or within a range of identities defined by any two of the aforementioned values across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion or within a range defined by any two of the aforementioned lengths) compared to a naturally occurring ROR2 protein.
  • the ROR2 protein is substantially identical to the protein identified by the UniProt reference number Q01974 (SEQ ID NO:10) or a variant or homolog having substantial identity thereto.
  • the ROR2 protein is substantially identical to the protein identified by the UniProt reference number Q9Z138 (SEQ ID NO:11) or a variant or homolog having substantial identity thereto.
  • siRNA small interfering RNA
  • small interfering RNA refer, in the usual and customary sense, to RNA molecules of typically 20-25 base pairs in length, which operate within the RNA interference pathway by interfering with the expression of specific genes with complementary nucleotide sequences. Without wishing to be bound by any theory, it is believed that siRNA assists in degradation of mRNA after transcription, thereby removing transcripts which would otherwise be expressed.
  • siRNA is specific for ROR1 or ROR2” means that the siRNA reduces or prevents expression of ROR1 or ROR2 upon contact in an expression system, e.g., in a cell.
  • a “Wnt5a protein” as referred to herein includes any of the recombinant or naturally-occurring forms of the wingless-type MMTV integration site family, member 5a (Wnt5a) or variants or homologs thereof that maintain Wnt5a activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Wnt5a or within a range of activity defined by any two of the aforementioned values compared to Wnt5a).
  • the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity or within a range of identities defined by any two of the aforementioned values across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion or within a range of defined by any two of the aforementioned lengths) compared to a naturally occurring Wnt5a protein.
  • the Wnt5a protein is substantially identical to the protein identified by the UniProt reference number P41221 (SEQ ID NO:12) or a variant or homolog having substantial identity thereto.
  • the Wnt5a protein is substantially identical to the protein identified by the UniProt reference number P22725 (SEQ ID NO:13) or a variant or homolog having substantial identity thereto.
  • RhoA protein or “RhoA” as referred to herein includes any of the recombinant or naturally-occurring forms of the Ras homolog gene family member A (RhoA) or variants or homologs thereof that maintain RhoA GTPase (guanosine triphosphatase) activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity or within a range of activity defined by any two of the aforementioned values compared to RhoA).
  • RhoA GTPase guanosine triphosphatase
  • the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity or within a range of identities defined by any two of the aforementioned values across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion or within a range of defined by any two of the aforementioned lengths) compared to a naturally occurring RhoA protein.
  • the RhoA protein is substantially identical to the protein identified by the UniProt reference number P61586 (SEQ ID NO:14) or a variant or homolog having substantial identity thereto.
  • the RhoA protein is substantially identical to the protein identified by the UniProt reference number Q9QUI0 (SEQ ID NO:15) or a variant or homolog having substantial identity thereto.
  • Ras-related C3 botulinum toxin substrate 1 Ras-related C3 botulinum toxin substrate 1
  • Rh1 GTPase guanosine triphosphatase
  • the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity or within a range of identities defined by any two of the aforementioned values across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion or within a range of lengths defined by any two of the aforementioned values) compared to a naturally occurring Rac1 protein.
  • the Rac1 protein is substantially identical to the protein identified by the UniProt reference number P63000 (SEQ ID NO:16) or a variant or homolog having substantial identity thereto.
  • the Rac1 protein is substantially identical to the protein identified by the UniProt reference number P63001 (SEQ ID NO:17) or a variant or homolog having substantial identity thereto.
  • a “ARHGEF1 protein” or “ARHGEF1” as referred to herein includes any of the recombinant or naturally-occurring forms of the Rho guanine nucleotide exchange factor 1 (ARHGEF1) or variants or homologs thereof that maintain ARHGEF1 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity or within a range of activity defined by any two of the aforementioned values compared to ARHGEF1).
  • ARHGEF1 Rho guanine nucleotide exchange factor 1
  • the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity or within a range of identities defined by any two of the aforementioned values across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion or within a range of lengths defined by any two of the aforementioned values) compared to a naturally occurring ARHGEF1 protein.
  • the ARHGEF1 protein is substantially identical to the protein identified by the UniProt reference number Q92888 (SEQ ID NO:18) or a variant or homolog having substantial identity thereto.
  • the ARHGEF1 protein is substantially identical to the protein identified by the UniProt reference number Q61210 (SEQ ID NO:19) or a variant or homolog having substantial identity thereto.
  • ARHGEF2 protein or “ARHGEF2” as referred to herein includes any of the recombinant or naturally-occurring forms of the Rho guanine nucleotide exchange factor 2 (ARHGEF2) or variants or homologs thereof that maintain ARHGEF2 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity or within a range of activity defined by any two of the aforementioned values compared to ARHGEF2).
  • ARHGEF2 Rho guanine nucleotide exchange factor 2
  • the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity or within a range of identities defined by any two of the aforementioned values across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion or within a range of lengths defined by any two of the aforementioned values) compared to a naturally occurring ARHGEF2 protein.
  • the ARHGEF2 protein is substantially identical to the protein identified by the UniProt reference number Q92974 (SEQ ID NO:20) or a variant or homolog having substantial identity thereto.
  • the ARHGEF2 protein is substantially identical to the protein identified by the UniProt reference number Q60875 (SEQ ID NO:21) or a variant or homolog having substantial identity thereto.
  • ARHGEF6 protein or “ARHGEF6” as referred to herein includes any of the recombinant or naturally-occurring forms of the Rho guanine nucleotide exchange factor 6 (ARHGEF6) or variants or homologs thereof that maintain ARHGEF6 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity or within a range of activity defined by any two of the aforementioned values compared to ARHGEF6).
  • ARHGEF6 Rho guanine nucleotide exchange factor 6
  • the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity or within a range of identities defined by any two of the aforementioned values across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion or within a range of lengths defined by any two of the aforementioned values) compared to a naturally occurring ARHGEF6 protein.
  • the ARHGEF6 protein is substantially identical to the protein identified by the UniProt reference number Q15052 (SEQ ID NO:22) or a variant or homolog having substantial identity thereto.
  • the ARHGEF6 protein is substantially identical to the protein identified by the UniProt reference number Q8K4I3 (SEQ ID NO:23) or a variant or homolog having substantial identity thereto.
  • UC-961 refers, in the usual and customary sense, to a monoclonal antibody drug agent which is capable of binding to ROR1, e.g., expressed on the surface of CLL cells. Binding of UC-961 to ROR1 may block cell growth and survival.
  • FRET fluorescence resonance energy transfer
  • two light-sensitive species e.g., detectable moieties such as chromophores or fluorophores.
  • Energy transfer between such molecules can be mediated through non-radiative dipole-dipole coupling, the efficiency of which is inversely proportional to the sixth-power of the distance.
  • FRET can be extremelyly sensitive to the distance between the donor and acceptor light-sensitive species, as known in the art.
  • KNG domain of ROR1 refers, in the usual and customary sense, to the extracellular kringle (KNG) domain of ROR1.
  • KNG domain of ROR2 refer, in the usual and customary sense, to the KNG domain of ROR2.
  • kringle domains are autonomous protein domains that fold into characteristic large loops stabilized by disulfide linkages.
  • the KNG domain corresponds to amino acid residues 310-392 of SEQ ID NO: 8.
  • the KNG domain corresponds to amino acid residues 314-395 of SEQ ID NO:10.
  • cancer refers to all types of cancer, neoplasm or malignant tumors found in mammals, including leukemias, lymphomas, melanomas, neuroendocrine tumors, carcinomas and sarcomas.
  • Exemplary cancers that may be treated with a compound, pharmaceutical composition, or method provided herein include lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g.
  • ER positive triple negative
  • ER negative chemotherapy resistant
  • herceptin resistant HER2 positive
  • doxorubicin resistant tamoxifen resistant
  • ductal carcinoma lobular carcinoma, primary, metastatic
  • ovarian cancer pancreatic cancer
  • liver cancer e.g. hepatocellular carcinoma
  • lung cancer e.g.
  • non-small cell lung carcinoma non-small cell lung carcinoma, squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma.
  • squamous cell carcinoma e.g., head, neck, or esophagus
  • colorectal cancer leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma.
  • Additional examples include, cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, esophagus, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus or Medulloblastoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial
  • leukemia refers broadly to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number abnormal cells in the blood-leukemic or aleukemic (subleukemic).
  • Exemplary leukemias that may be treated with a compound, pharmaceutical composition, or method provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous
  • sarcoma generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
  • Sarcomas that may be treated with a compound, pharmaceutical composition, or method provided herein include a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sar
  • melanoma is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
  • Melanomas that may be treated with a compound, pharmaceutical composition, or method provided herein include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma.
  • carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • exemplary carcinomas that may be treated with a compound, pharmaceutical composition, or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma
  • the terms “metastasis,” “metastatic,” and “metastatic cancer” can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or originating site acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and/or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites and tissues in the body.
  • a second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor.
  • the metastatic tumor and its cells are presumed to be similar to those of the original tumor.
  • the secondary tumor in the breast is referred to a metastatic lung cancer.
  • metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors.
  • non-metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors.
  • metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e.g., in the breast.
  • a disease e.g., diabetes, cancer (e.g. prostate cancer, renal cancer, metastatic cancer, melanoma, castration-resistant prostate cancer, breast cancer, triple negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma)) means that the disease (e.g., diabetes, cancer (e.g. prostate cancer, renal cancer, metastatic cancer, melanoma, castration-resistant prostate cancer, breast cancer, triple negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myel
  • lung cancer ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma) is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function.
  • skin cancer e.g., Merkel cell carcinoma
  • testicular cancer e.g., leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma
  • a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function.
  • ROR1-associated proteins in primary CLL cells were examined. It was unexpectedly found that ROR1 couples with ROR2 in response to Wnt5a to recruit guanine exchange factors (GEFs) that activate Rho GTPases.
  • GEFs guanine exchange factors
  • Wnt5a can activate GTPases RhoA and Rac1 in chronic lymphocytic leukemia (CLL) cells to enhance leukemia-cell migration and proliferation, respectively. It has been found that Wnt5a is present at high levels in the plasma of patients with CLL relative to that of healthy donors and that of freshly isolated leukemia cells.
  • CLL chronic lymphocytic leukemia
  • a method of determining an expression level of a Wnt5a protein in a subject that has or is at risk for developing cancer includes (i) obtaining a biological sample from the subject; (ii) determining an expression level of a Wnt5a protein in the biological sample.
  • the determining includes: (a) contacting the Wnt5a protein with a Wnt5a protein binding agent in the biological sample, thereby forming a Wnt5a protein-binding agent complex; and (b) detecting said Wnt5a protein-binding agent complex.
  • Wnt5a protein binding agent refers to a substance capable of binding a Wnt5a protein.
  • the Wnt5a protein binding agent may be a nucleic acid or a protein.
  • the Wnt5a protein binding agent is an aptamer.
  • the Wnt5a protein binding agent is peptide.
  • the Wnt5a protein binding agent is a small molecule.
  • the Wnt5a protein binding agent is an antibody.
  • the Wnt5a protein binding agent includes a detectable moiety.
  • the method includes further selecting a subject that has or is at risk for developing cancer.
  • the biological sample is a blood-derived biological sample of the subject.
  • the blood-derived biological sample is whole blood, serum or plasma.
  • the cancer is chronic lymphocytic leukemia (CLL).
  • ROR1-associated proteins in chronic lymphocytic leukemia can be found on CLL and CD5 B cells.
  • Wnt5a induces ROR1-ROR2 coupling (binding), which recruits ARHGEF1, ARHGEF2 and ARHGEF6, and activated RhoA and Rac1, enhancing CLL-cell migration and proliferation.
  • CLL-cell-line MEC1 which lacks ROR1 but expresses ROR2 and Wnt5a
  • MEC1 made to express ROR1 had ROR1-ROR2 complexes, which recruited guanine exchange factors, increased cytokine-directed migration, and enhanced engraftment in immune-deficient mice.
  • the extracellular Kringle domain is required for ROR1-ROR2 coupling (binding) and the cysteine-rich domain or intracellular proline-rich domain is required for Wnt5a-induced recruitment of guanine exchange factors to ROR1-ROR2.
  • coupling (binding) of ROR1 with ROR2 accounts for its noted biologic activity in blocking non-canonical Wnt signaling by Wnt5a, which, it has been found, enhances the proliferation, survival, and metastasis of cancer cells in vivo.
  • antibodies can inhibit the capacity of ROR1 to couple with ROR2, and thereby block the effect that Wnt5a can have on cancer cells in inducing activation of RhoA and Rac1.
  • ROR1-ROR2 coupling (binding) inhibitors may be performed with fluorescence confocal microscopy or Fluorescence Resonance Energy Transfer (FRET) assay to monitor for co-localization of ROR1 with ROR2 or by other means, such as a cell-based assay. Screening could be automated so that it could be used to screen for antibodies or other agents (small molecules, peptides, drugs, etc.), which interfere with the coupling (binding) of ROR1 and ROR2. Agents that interfere with such coupling (binding) would be expected to interfere with the non-canonical Wnt signaling induced by Wnt5a.
  • FRET Fluorescence Resonance Energy Transfer
  • identifying agents capable of inhibiting the binding between ROR1 and ROR2 (e.g., the formation or maintenance of a protein complex comprising ROR1 and ROR2).
  • the identified agents may inhibit ROR1-ROR2-binding and/or the formation or maintenance of a protein complex comprising ROR1 and ROR2 by interacting with ROR1, thereby preventing binding of ROR1 to ROR2.
  • the identified agents may inhibit the binding by interacting with ROR2, thereby preventing binding of ROR1 to ROR2.
  • the agents identified by the methods provided herein may be useful as cancer diagnostics or cancer therapeutics.
  • a method of identifying an inhibitor of ROR1-ROR2 binding and/or the formation or maintenance of a protein complex comprising ROR1 and ROR2 includes (i) combining a test agent with a ROR1 protein and a ROR2 protein in a reaction vessel.
  • a method of identifying an inhibitor of ROR1-ROR2 binding includes (i) combining a test agent with a ROR1 protein and a ROR2 protein in a reaction vessel. (ii) A decrease in binding of the ROR1 protein to the ROR2 protein relative to a standard control is detected and thereby an inhibitor of ROR1-ROR2 binding is identified.
  • binding refers to the association between a ROR1 protein and a ROR2 protein. The association can be direct (e.g., by covalent bond) or indirect (e.g., by non-covalent bond (e.g. electrostatic interactions (e.g.
  • binding includes the formation or maintenance of a protein complex comprising ROR1 and ROR2.
  • ROR1-ROR2 binding and/or the formation or maintenance of a protein complex comprising ROR1 and ROR2 may also be referred to as “heterooligomerization” or “coupling.”
  • ROR1-ROR2 binding and/or the formation or maintenance of a protein complex comprising ROR1 and ROR2 is preceded by binding of Wnt5a to ROR1 or ROR2.
  • ROR1-ROR2 binding and/or the formation or maintenance of a protein complex comprising ROR1 and ROR2 increases the level of a guanine exchange factor (GEF) or a GTPase.
  • GEF guanine exchange factor
  • the interaction between the ROR1 protein and the ROR2 protein and/or the formation or maintenance of a protein complex comprising ROR1 and ROR2 may be inhibited by binding of the test agent to a KNG domain.
  • the KNG domain forms part of the ROR1 protein.
  • the KNG domain forms part of the ROR2 protein.
  • the ROR1 protein and the ROR2 protein may be expressed by a cell.
  • the reaction vessel includes a cell.
  • the ROR1 protein and the ROR2 protein are expressed on the surface of a cell.
  • the cell may form part of an in vitro cell culture.
  • the in vitro cell culture includes a Wnt5 protein.
  • the combining step occurs in the presence of a Wnt5a protein.
  • the cell expresses an endogenous Wnt5a protein.
  • the formation or maintenance of a protein complex comprising ROR1 and ROR2 occurs in the presence of a Wnt5a protein.
  • the cell expresses an exogenous Wnt5a protein.
  • the cell is transfected with a vector encoding a Wnt5a protein.
  • the reaction vessel includes a cell culture.
  • a “cell culture” as provided herein refers to an environment including appropriate cellular nutrients and capable of maintaining cells in vitro.
  • the environment may be a liquid environment, a solid environment and/or a semisolid environment (e.g. agar, gel etc.) in an appropriate vessel (e.g., cell culture dish).
  • a cell culture medium may be employed.
  • a “cell culture medium” as used herein, is used according to its generally accepted meaning in the art.
  • a cell culture medium (also referred to in the art and herein as a “culture medium”) includes liquids (e.g., growth factors, minerals, vitamins etc.) or gels designed to support the growth (e.g. division, differentiation, maintenance etc.) of cells.
  • compositions provided herein including embodiments further include a physiologically acceptable solution.
  • a “physiologically acceptable solution” as provided herein refers to any acceptable aqueous solution (e.g., buffer) in which the compositions provided herein may be contained without losing their biological properties.
  • the physiologically acceptable solution is a cell culture medium.
  • the cell forms part of an in vitro cell culture.
  • the cell is a cancer cell.
  • the cancer cell is a chronic lymphocytic leukemia (CLL) cell.
  • the CLL cell is a chronic B cell leukemia cell.
  • the CLL cell is a MEC1 cell.
  • a MEC1 cell as provided herein refers to a cell as deposited with the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures and having the accession number ACC 497.
  • the MEC1 cell expresses a ROR1 protein and a ROR2 protein.
  • the ROR1 protein is an exogenous protein and the ROR2 protein is an endogenous protein.
  • the MEC1 cell may endogenously express a ROR2 protein and by transfection with a vector expressing a ROR1 protein form a MEC1 cell expressing a ROR1 protein and a ROR2 protein.
  • the ROR1 protein and the ROR2 protein are isolated proteins and the method is performed in a cell-free environment.
  • the combining includes binding (allowing binding of) an isolated ROR1 protein to a solid support thereby forming a bound ROR1 protein and contacting the bound ROR1 protein with an isolated ROR2 protein and a test agent.
  • the reaction vessel is a column including a solid support.
  • a “solid support” as provided herein refers to any material that can be modified to contain discrete individual sites appropriate for the attachment or association of an isolated ROR1 protein or an isolated ROR2 protein or fragment thereof as provided herein including embodiments thereof and is amenable to the methods provided herein including embodiments thereof.
  • solid supports include without limitation, glass and modified or functionalized glass (e.g., carboxymethyldextran functionalized glass), plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonTM, etc.), polysaccharides (e.g., sepharose, agarose), nylon or nitrocellulose, composite materials, ceramics, and plastic resins, silica or silica-based materials including silicon and modified silicon (e.g., patterned silicon), carbon, metals, quartz (e.g., patterned quartz), inorganic glasses, plastics, optical fiber bundles, and a variety of other polymers.
  • the substrates allow optical detection and do not appreciably fluoresce.
  • the detecting includes detecting a level of the ROR1 protein bound to the ROR2 protein in the presence of the test agent, wherein a decreased level of the ROR1 protein bound to the ROR2 protein relative to a standard control indicates the test agent is an inhibitor of ROR1-ROR2 binding.
  • the standard control may be the level of binding of a ROR1 protein to a ROR2 protein in the absence of the test agent.
  • the standard control is the level of binding of a ROR1 protein to a ROR2 protein in the presence of an agent unable to inhibit binding of the ROR1 protein to the ROR2 protein.
  • the standard control is the level of binding of a ROR1 protein to a ROR2 protein in the presence of an agent unable to bind to the ROR1 protein or the ROR2 protein. In embodiments, the standard control is the level of binding of a ROR1 protein to a ROR2 protein in the presence of an agent unable to bind to the KNG domain of a ROR1 protein. In embodiments, the standard control is the level of binding of a ROR1 protein to a ROR2 protein in the presence of a ROR1 antibody. In further embodiments, said ROR1 antibody is a 4A5 antibody.
  • the detecting includes detecting a level of the formation or maintenance of a protein complex comprising ROR1 and ROR2 in the presence of the test agent, wherein a decreased level of the ROR1 protein or the ROR2 protein in the complex relative to a standard control indicates the test agent is an inhibitor of ROR1-ROR2 binding.
  • the standard control may be the level of the formation or maintenance of a protein complex comprising ROR1 and ROR2 in the absence of the test agent.
  • the standard control is the formation or maintenance of a protein complex comprising ROR1 and ROR2 in the presence of an agent previously shown to not inhibit binding of the ROR1 protein to the ROR2 protein.
  • the standard control is the level of the formation or maintenance of a protein complex comprising ROR1 and ROR2 in the presence of an agent previously shown to not bind to the ROR1 protein or the ROR2 protein. In embodiments, the standard control is the level of the formation or maintenance of a protein complex comprising ROR1 and ROR2 in the presence of an agent previously shown to not bind to the KNG domain of a ROR1 protein. In embodiments, the standard control is the level of the formation or maintenance of a protein complex comprising ROR1 and ROR2 in the presence of a ROR1 antibody. In further embodiments, said ROR1 antibody is a 4A5 antibody.
  • the detecting includes detecting a level of unbound ROR1 protein or unbound ROR2 protein in the presence of the test agent, wherein an increased level of unbound ROR1 protein or an increased level of unbound ROR2 protein relative to a standard control indicates the test agent is an inhibitor of ROR1-ROR2 binding.
  • unbound ROR1 protein refers to a ROR1 protein, which does not form part of a complex or is not bound to a ROR2 protein.
  • unbound ROR2 protein refers to a ROR2 protein, which does not form part of a complex or is not bound to a ROR1 protein.
  • the standard control is the level of unbound ROR1 protein or unbound ROR2 protein in the absence of the test agent. In embodiments, the standard control is the level of unbound ROR1 protein or unbound ROR2 protein in the presence of an agent unable to inhibit binding of the ROR1 protein to the ROR2 protein. In embodiments, the standard control is the level of unbound ROR1 protein or unbound ROR2 protein in the presence of an agent unable to bind to the ROR1 protein or the ROR2 protein. In embodiments, the standard control is the level of binding of a ROR1 protein to a ROR2 protein in the presence of an agent unable to bind to the KNG domain of a ROR1 protein. In embodiments, the standard control is the level of binding of a ROR1 protein to a ROR2 protein in the presence of a ROR1 antibody. In further embodiments, said ROR1 antibody is a 4A5 antibody.
  • the detecting includes detecting a level of unbound ROR1 protein or unbound ROR2 protein in a protein complex comprising ROR1 and ROR2 in the presence of the test agent, wherein an increased level of unbound ROR1 protein or an increased level of unbound ROR2 protein relative to a standard control indicates the test agent is an inhibitor of the formation or maintenance of a protein complex comprising ROR1 and ROR2.
  • the standard control is the level of unbound ROR1 protein or unbound ROR2 protein in a protein complex comprising ROR1 and ROR2 in the absence of the test agent.
  • the standard control is the level of unbound ROR1 protein or unbound ROR2 protein in a protein complex comprising ROR1 and ROR2 in the presence of an agent previously shown to not inhibit the formation or maintenance of a protein complex comprising ROR1 and ROR2.
  • the standard control is the level of the formation or maintenance of a protein complex comprising ROR1 and ROR2 in the presence of an agent previously shown to not bind to the KNG domain of a ROR1 protein.
  • the standard control is the level of binding of the formation or maintenance of a protein complex comprising ROR1 and ROR2 in the presence of a ROR1 antibody.
  • said ROR1 antibody is a 4A5 antibody.
  • the detecting includes detecting a level of a guanine exchange factor (GEF) protein activity or a guanosine triphosphatase (GTPase) protein activity in the presence of the test agent, wherein a decreased level of the GEF protein activity or the GTPase protein activity relative to a standard control indicates the test agent is an inhibitor of ROR1-ROR2 binding (e.g., the formation or maintenance of a protein complex comprising ROR1 and ROR2).
  • the standard control is the level of GEF protein activity or GTPase protein activity in the absence of the test agent.
  • the GEF protein is ARHGEF1, ARHGEF2 or ARHGEF6.
  • the GEF protein is ARHGEF1.
  • the GEF protein is ARHGEF2. In embodiments, the GEF protein is ARHGEF6. In embodiments, the GTPase protein is RhoA or Rac1. In embodiments, the GTPase protein is RhoA. In embodiments, the GTPase protein is Rac1.
  • the ROR1 protein and the ROR2 protein include a detectable moiety.
  • the GEF protein or the GTPase protein include a detectable moiety.
  • a detectable moiety is as defined herein.
  • the detectable moiety is a bioluminescent molecule.
  • the detectable moiety is a photoactive molecule.
  • the detectable moiety is a fluorophore.
  • the detectable moiety is an antibody including a fluorophore.
  • the detectable moiety and the ROR1 protein form a fusion protein.
  • the detectable moiety and the ROR2 protein form a fusion protein.
  • detectable moieties can be used, with the choice of label depending on the sensitivity required, ease of conjugation with the antibody, stability requirements, and available instrumentation and disposal provisions.
  • Suitable detectable moieties include, but are not limited to, radionuclides, fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon GreenTM, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc.), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), autoquenched fluorescent compounds that are activated by tumor-associated proteases, enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, and the like.
  • the detectable moiety is amenable to a Fluorescence Reson
  • the step of detection as provided herein may include immunoassay techniques and protocols generally known and described in the art. See Price and Newman, “Principles and Practice of Immunoassay,” 2nd Edition, Grove's Dictionaries, 1997; and Gosling, “Immunoassays: A Practical Approach,” Oxford University Press, 2000. A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used (see, e.g., Self et al., Curr. Opin. Biotechnol., 7:60-65 (1996)).
  • immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (META); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA); and chemiluminescence assays (CL). If desired, such immunoassays can be automated.
  • EIA enzyme multiplied immunoassay technique
  • ELISA enzyme-linked immunosorbent assay
  • MAC ELISA IgM antibody capture ELISA
  • MEA microparticle enzyme immunoassay
  • CEIA capillary electrophoresis immunoassays
  • RIA radioimmunoassays
  • IRMA immunoradio
  • Immunoassays can also be used in conjunction with laser induced fluorescence (see, e.g., Schmalzing et al., Electrophoresis, 18:2184-93 (1997); Bao, J. Chromatogr. B. Biomed. Sci., 699:463-80 (1997)).
  • Liposome immunoassays such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention (see, e.g., Rongen et al., J. Immunol. Methods, 204:105-133 (1997)).
  • nephelometry assays in which the formation of protein/antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the marker concentration, are suitable for use in the methods of the present invention.
  • Nephelometry assays are commercially available from Beckman Coulter (Brea, Calif.; Kit #449430) and can be performed using a Behring Nephelometer Analyzer (Fink et al., J. Clin. Chem. Clin. Biochem., 27:261-276 (1989)).
  • Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody.
  • An antibody labeled with iodine-125 ( 125 I) can be used.
  • a chemiluminescence assay using a chemiluminescent antibody specific for the protein marker is suitable for sensitive, non-radioactive detection of protein levels.
  • An antibody labeled with fluorochrome is also suitable.
  • fluorochromes examples include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and lissamine.
  • Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), ⁇ -galactosidase, urease, and the like.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • AP alkaline phosphatase
  • ⁇ -galactosidase urease
  • a horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm.
  • An alkaline phosphatase detection system can be used with the chromogenic substrate p-nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm.
  • a ⁇ -galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl- ⁇ -D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm.
  • An urease detection system can be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals; St. Louis, Mo.).
  • a signal from the direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of 125 I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength.
  • a quantitative analysis can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, Calif.) in accordance with the manufacturer's instructions.
  • the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.
  • the detecting step includes detecting ROR1-ROR2 binding by confocal microscopy.
  • Optical images viewed (and, optionally, recorded) by a camera or other recording device are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and storing and analyzing the image on a computer.
  • a camera or other recording device e.g., a photodiode and data storage device
  • a variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical images.
  • One conventional system carries light from the specimen field to a cooled charge-coupled device (CCD) camera, in common use in the art.
  • a CCD camera includes an array of picture elements (pixels). The light from the specimen is imaged on the CCD. Particular pixels corresponding to regions of the specimen are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed.
  • the apparatus and methods of the invention are easily used for viewing any sample, e.g., by fluorescent or dark field microscopic techniques.
  • test agents provided herein may be any compound capable of inhibiting the binding of a ROR1 protein to a ROR2 protein (e.g., the formation or maintenance of a protein complex comprising ROR1 and ROR2).
  • the test agent is an antibody, a small molecule, a peptide, a protein or a nucleic acid.
  • the test agent is an antibody.
  • the antibody may be an anti-ROR1 antibody.
  • the antibody is an anti-ROR2 antibody.
  • the antibody binds to a ROR1 protein or a ROR2 protein.
  • the antibody binds to a KNG domain.
  • the antibody is a humanized antibody.
  • the antibody is a chimeric antibody. In embodiments, the antibody is a scFv. In embodiments, the test agent is a small molecule. In embodiments, the test agent is a peptide. In embodiments, the test agent is a protein. In embodiments, the test agent is a nucleic acid.
  • an antibody identified by the method provided herein including embodiments thereof is provided.
  • the antibody is an anti-ROR1 antibody.
  • the antibody binds the KNG domain of a ROR1 protein.
  • the antibody is cirmtuzumab.
  • a method of inhibiting a ROR1-ROR2 interaction (e.g., the formation or maintenance of a protein complex comprising ROR1 and ROR2) is provided.
  • the method includes (i) contacting a compound identified by the method provided herein including embodiments thereof with a ROR1-ROR2 complex, wherein the complex includes a ROR1 protein and a ROR2 protein. (ii) The compound is allowed to inhibit the interaction between the ROR1 protein and the ROR2 protein, thereby inhibiting a ROR1-ROR2 interaction (e.g., the formation or maintenance of a protein complex comprising ROR1 and ROR2).
  • the agents identified herewith may be, inter alia, useful for the treatment of cancer.
  • a method of treating cancer in a subject in need thereof includes administering to a subject a therapeutically effective amount of an antibody, small molecule, peptide or nucleic acid identified by a method provided herein including embodiments thereof.
  • “Patient” or “subject in need thereof” refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a composition or pharmaceutical composition as provided herein.
  • Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals.
  • a patient is human.
  • the terms “disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with a compound, pharmaceutical composition, or method provided herein.
  • the disease is cancer (e.g. lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma).
  • cancer e.g. lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma).
  • treating refers to any indicia of success in the treatment or amelioration of an injury, disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient's physical or mental well-being.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric exams, and/or a psychiatric evaluation.
  • the term “treating” and conjugations thereof, include prevention of an injury, pathology, condition, or disease.
  • “treating” refers to treatment of cancer.
  • an “effective amount” is an amount sufficient for a compound to accomplish a stated purpose relative to the absence of the compound (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signaling pathway, or reduce one or more symptoms of a disease or condition).
  • An example of an “therapeutically effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.”
  • a “reduction” of a symptom or symptoms means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).
  • “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient.
  • Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like.
  • Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances, and the like., that do not deleteriously react with the compounds of the invention.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances, and the like.
  • preparation is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it.
  • carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it.
  • cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
  • administering means oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject.
  • Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).
  • Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.
  • Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.
  • co-administer it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies.
  • the compound of the invention can be administered alone or can be co-administered to the patient.
  • Co-administration is meant to include simultaneous or sequential administration of the compound individually or in combination (more than one compound or agent).
  • the preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation).
  • compositions disclosed herein can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
  • Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions.
  • the compositions of the present invention may additionally include components to provide sustained release and/or comfort.
  • Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes.
  • the compositions disclosed herein can also be delivered as microspheres for slow release in the body.
  • microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997).
  • the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis.
  • liposomes particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo.
  • the compositions can also be delivered as nanoparticles.
  • compositions may include compositions wherein the active ingredient (e.g. compounds described herein, including embodiments or examples) is contained in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose.
  • a therapeutically effective amount i.e., in an amount effective to achieve its intended purpose.
  • the actual amount effective for a particular application will depend, inter alia, on the condition being treated.
  • compositions When administered in methods to treat a disease, such compositions will contain an amount of active ingredient effective to achieve the desired result, e.g., modulating the activity of a target molecule, and/or reducing, eliminating, or slowing the progression of disease symptoms.
  • the dosage and frequency (single or multiple doses) administered to a mammal can vary depending upon a variety of factors, for example, whether the mammal suffers from another disease, and its route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of symptoms of the disease being treated, kind of concurrent treatment, complications from the disease being treated or other health-related problems.
  • Other therapeutic regimens or agents can be used in conjunction with the methods and compounds of Applicants' invention. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the ability of those skilled in the art.
  • the therapeutically effective amount can be initially determined from cell culture assays.
  • Target concentrations will be those concentrations of active compound(s) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art.
  • therapeutically effective amounts for use in humans can also be determined from animal models.
  • a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals.
  • the dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.
  • Dosages may be varied depending upon the requirements of the patient and the compound being employed.
  • the dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.
  • CLL cells express ROR2 coupled with ROR1, apparently in response to Wnt5a, which we found present at relatively high levels in the plasma of patients with CLL.
  • ROR1-ROR2 complex dissociated, but could be re-formed by treatment with Wnt5a, which induced recruitment of GEFs and the activation of RhoA and Rac1.
  • RhoA was associated with enhanced chemokine-induced migration
  • Rac1 was associated with enhanced leukaemia-cell proliferation in response to membrane-bound CD154 and IL-4/10.
  • KNG domains contain intra-domain disulfide bridges, which define polypeptide loops that often are involved in protein-protein interactions 34 .
  • the KNG domain of ROR1, and possibly ROR2 may interact with one another to form a heterodimeric complex in response to Wnt5a (e.g. FIG. 5I ).
  • Wnt5a e.g. FIG. 5I
  • the failure of the ⁇ KNG-ROR1 to couple with ROR2 also could be due to steric constraints introduced by truncation of the ROR1 extracellular domain, ⁇ CRD-ROR1, lacking the larger extra-cellular CRD, could couple with the ROR2.
  • Wnt5a binding to ROR2 may be sufficient to allow for ROR2 to couple with ROR1.
  • the CRD may have residues that ordinarily inhibit such coupling unless bound to Wnt5a.
  • neither ⁇ CRD-ROR1 nor any one of the other truncated forms of ROR1 allowed for recruitment of GEFs to ROR1-ROR2 or enhanced activation of RhoA and Rac1 in transfected MEC1 cells.
  • ROR1-ROR2 Because each of the truncated forms of ROR1, except for ⁇ KNG-ROR1, allowed for ROR1 to complex with ROR2, the coupling of ROR1 with ROR2 does not appear sufficient for Wnt5a-induced non-canonical signaling. Moreover, the intracellular domains of ROR1 appear necessary, including the proline-rich domain (PRD), which contains several putative SH3-binding sites, which may permit docking of GEFs and/or scaffolding proteins to the ROR1-ROR2 complex.
  • PRD proline-rich domain
  • cells may require co-expression of both ROR1 and ROR2 to trigger optimal Wnt5a-induced activation of RhoA and Rac1.
  • Wnt5a instead may induce ROR2 to bind other Frizzled (Fzd) receptors 37-39 which otherwise couple with LRP5/6 in response to canonical Wnt factors, such as Wnt3a 27,32,38,39 .
  • ROR2/Fzd complexes may compete with the formation of LPR/Fzd complexes, thereby inhibiting the capacity of other Wnt factors to induce canonical Wnt signaling 27,32,38,39 .
  • tumour/growth-suppressors may depend in part upon the relative expression of ROR1, LPR5/6, and Fzd receptors, which can couple with ROR2 in response to Wnt5a.
  • ROR2 appears more widely expressed on post-partum tissues than ROR1, which appears confined to a rare subpopulation of precursor B cells, called hematogones 14 .
  • ROR2 on normal CD5 B cells, which are the presumed normal counterpart to CLL40.
  • acquisition of ROR1 by such cells may be an important step in leukaemogenesis, allowing the neoplastic B cells to activate RhoA and Rac1 in response to Wnt5a, which we found at relatively high-levels in the plasma of patients with CLL.
  • CLL cells can be induced to proliferate upon culture with cells expressing CD154 (HeLa CD154 ) in the presence of exogenous interleukin (IL)-4 and IL-10 ( FIG. 1 a, b ).
  • IL interleukin
  • Wnt5a significantly enhanced the proportion of dividing cells and the numbers of cell divisions that could be deduced from the fluorescence of cells labeled with carboxyfluorescein succinimidyl ester (CFSE); the enhanced proliferation induced by Wnt5a could be inhibited by an anti-ROR1 mAb (UC-961) to levels comparable to those observed in cultures without Wnt5a ( FIG. 1 a, b ).
  • CLL cells co-cultured with HeLa cells were not induced to proliferate, even in the presence of IL-4/10 and/or Wnt5a ( FIG. 7 ).
  • exogenous Wnt5a also could enhance migration of CLL cells toward chemokines, e.g. CXCL12 ( FIG. 1 c ) [41].
  • the capacity of Wnt5a to enhance migration could be inhibited by UC-961.
  • exogenous Wnt5a without CXCL12 did not induce CLL-cell migration, and UC-961 did not inhibit the migration of CLL cells to CXCL12 without Wnt5a ( FIG. 1 c ).
  • Rho family proteins play important roles in regulating proliferation and/or migration [42], and Wnt5a has been reported to activate Rac1 and RhoA in other cell types [38,43].
  • Wnt5a could induce activation of Rac1 and RhoA within 30 min ( FIG. 1 d ).
  • Addition of UC-961 could inhibit Wnt5a-induced activation of Rac1 and RhoA ( FIG. 1 e ).
  • CXCL12 activated RhoA in CLL cells ( FIG. 1 g ) [44].
  • exogenous Wnt5a enhanced the level of Rac1 or RhoA activated by CD154 or CXCL12, respectively ( FIG. 1 f, g ).
  • NSC-23766 an inhibitor of Rac1 GTPase, but not Y-27632, a selective inhibitor of p160ROCK, could inhibit the proliferation induced by HeLa CD154 , with or without exogenous Wnt5a.
  • Y-27632, but not NSC-23766 could inhibit chemotaxis to CXCL12, with or without exogenous Wnt5a, supporting the notion that activation of Rac1 or RhoA can promote CLL-cell proliferation or migration, respectively ( FIG. 1 h - j ).
  • CD19 + blood B cells of healthy adults also expressed ROR2, including B cells that co-expressed CD5 ( FIG. 2 c ).
  • ROR2 mean-fluorescence intensity
  • FIGS. 10A-10B Transfecting CLL cells with siRNA specific for ROR1 or ROR2, but not control siRNA, respectively lowered expression of only ROR1 or ROR2 by immunoblot analysis or flow cytometry.
  • GEFs can activate RhoA and Rac1 [45].
  • Mass spectrometry analysis detected ARHGEF1, ARHGEF2, and ARHGEF6 in the anti-ROR1 ip ( FIG. 11A ). This association was confirmed by immunoblot analysis of the anti-ROR1 ip, which we found contained each of these GEFs ( FIG. 11B ).
  • the ip generated from lysates of freshly-isolated CLL cells using mAbs specific for either ARHGEF1, ARHGEF2, or ARHGEF6 each contained ROR1 detectable by immunoblot analysis ( FIG. 11B ).
  • Fluorescence confocal microscopy showed that ROR1 and ROR2 co-localized with ARHGEF1, ARHGEF2, or ARHGEF6 in cultured CLL cells that were treated with exogenous Wnt5a ( FIG. 4 a - c ). However, treatment with UC-961 inhibited the capacity of Wnt5a to induce recruitment of any one of these GEFs to either ROR1 or ROR2 ( FIG. 12A ).
  • treatment with Wnt5a increased the in vitro exchange activity for RhoA of ip generated with mAbs specific for either ARHGEF1 or ARHGEF2, but not ARHGEF6.
  • treatment with Wnt5a increased the in vitro exchange activity for Rac1 using ip generated with mAbs specific for either ARHGEF2 or ARHGEF6, but not ARHGEF1 ( FIGS. 4D and 12B ).
  • Treatment with UC-961 inhibited the capacity of Wnt5a to induce activation of RhoA by ip generated from anti-ARHGEF1 or anti-ARHGEF2, or Rac1 by ip of anti-ARHGEF2 or anti-ARHGEF6, respectively ( FIG. 4 e ).
  • Wnt5a was less effective in activating RhoA in CLL cells transfected with siRNA specific for either ARHGEF1 or ARHGEF2 than in CLL cells transfected with control siRNA ( FIGS. 4F and 12C ).
  • Wnt5a was less effective in activating Rac1 in CLL cells transfected with siRNA specific for either ARHGEF2 or ARHGEF6 ( FIGS. 4F and 12C ).
  • CLL cells silenced for ARHGEF1 or ARHGEF6 respectively did not have impaired Wnt5a-induced activation of Rac1 or RhoA ( FIG. 12D ), indicating that ARHGEF1 and ARHGEF2, or ARHGEF2 and ARHGEF6, were required for optimal Wnt5a-induced activation of RhoA or Rac1, respectively.
  • MEC1 cells were derived from CLL and have been used as a cell-model for this leukaemia [30]. However, we found that MEC1 cells lacked ROR1, but did express ROR2 ( FIG. 13A ). Stable transfection of MEC1 with vectors encoding full-length ROR1, or each of various truncated-forms of ROR1 lacking distinct structural domains, allowed us to generate MEC1 that expressed high-levels of surface ROR1, which could be detected via flow cytometry using 4A5 mAb ( FIG. 13A ). Expression of ROR1, or any one of the various truncated forms of ROR1, did not alter the expression-level of surface ROR2 ( FIG. 13A ).
  • MEC1 cells express high levels of Wnt5a ( FIG. 13B ) [41], we assessed for constitutive ROR1-ROR2 coupling in MEC1 cells made to express ROR1.
  • Fluorescence confocal microscopy demonstrated that ROR1 and ROR2 co-localized with ARHGEF1, ARHGEF2, or ARHGEF6 in MEC1-ROR1 cells ( FIG. 5 b ).
  • MEC1-ROR1 cells that expressed any of the truncated forms of ROR1 ( FIGS. 14B-14C ).
  • MEC1-ROR1 cells also had higher levels of activated Rac1 and RhoA than MEC1 ( FIG. 5 c ); such enhanced activation could be inhibited by treatment with either UC-961 or neutralizing antibodies to Wnt5a ( FIG. 5 d ).
  • the MEC1 cells transfected with any one of the various truncated forms of ROR1 did not have levels of activated Rac1 or RhoA that were greater than that of the parental MEC1 cells.
  • MEC1-ROR1 cells grew faster than parental MEC1 cells or MEC1 cells transfected with any one of the truncated forms of ROR1 ( FIG. 5 e ).
  • Treatment with either neutralizing antibodies to Wnt5a or UC-961 could inhibit the growth of MEC1-ROR1 cells (FIG. 5 f ).
  • MEC1-ROR1 migrated significantly better in response to CCL21 than did MEC1 cells, or MEC1 expressing any of the truncated forms of ROR1 ( FIG. 5 g ).
  • MEC1 cells could engraft immune-deficient mice [46].
  • treatment with UC-961 significantly inhibited engraftment of MEC1-ROR1 ( FIG. 6 c - e ).
  • the MEC1-ROR1 cells harvested from mice treated with UC-961 had lost or attenuated their expression of ROR1 ( FIG. 6 d ).
  • Embodiments contemplated herein include the following.
  • Embodiment P1 A method for identifying an agent that disrupts ROR1 and ROR2 coupling comprising: a. Contacting CLL cells co-expressing ROR1 and ROR2 with Wnt5a; b. contacting said CLL cells with the agent or a control; and c. determining the presence or absence of ROR1 and ROR2 coupling, wherein the absence of ROR1 and ROR2 coupling in the agent contacted cells compared to the control cells is indicative of disruption of ROR1 and ROR2 coupling, by which an agent that disrupts ROR1 and ROR2 coupling is identified.
  • Embodiment P2 The method of embodiment P1, wherein the agent is an antibody, a peptide, a small molecule or siRNA.
  • Embodiment P3 The method of embodiment P1 or P2, wherein the agent is an antibody.
  • Embodiment P4 The method of one of embodiments P1-P3, wherein the antibody is UC-961.
  • Embodiment P5. The method of one of embodiments P1-P4, wherein the agent is a siRNA.
  • Embodiment P6 The method of embodiment P5, wherein the siRNA is specific for ROR1 or ROR2.
  • Embodiment P7 The method one of embodiments P1-P6, wherein the presence or absence of ROR1 and ROR2 coupling is determined by measuring co-localization of ROR1 and ROR2 or by inhibition of Wnt5a activity.
  • Embodiment P8 The method of embodiment P7, wherein the co-localization of ROR1 and ROR2 or by inhibition of Wnt5a activity is determined using a fluorescence assay.
  • Embodiment P9 The method of embodiment P8, wherein the fluorescence assay is selected from the group consisting of: fluorescence confocal microscopy, fluorescence resonance energy transfer (FRET), ELISA, FACS, mass spectroscopy, and cell based assay.
  • FRET fluorescence resonance energy transfer
  • ELISA fluorescence confocal microscopy
  • FACS fluorescence resonance energy transfer
  • FACS mass spectroscopy
  • cell based assay cell based assay.
  • Embodiment P10 The method of embodiment P7, wherein inhibition of Wnt5a activity is indicated by decreased or lack of RhoA and RAC1 activity in the agent contacted cells compared with the control cells.
  • Embodiment P11 The method of embodiment P7, wherein inhibition of Wnt5a activity is indicated by decreased or lack of ARHGEF1, ARHGEF2 and/or ARHGEF6 expression in the agent contacted cells compared with the control cells.
  • Embodiment P12 The method of one of embodiments P1-P11, wherein the CLL cells are MEC1 cells.
  • Embodiment P13 An isolated antibody, which inhibits ROR1 and ROR2 coupling.
  • Embodiment P14 The antibody of embodiment P13, wherein the antibody is monoclonal, chimeric, human or humanized.
  • Embodiment P15 The antibody of embodiment P13, wherein the antibody inhibits Wnt5a activity.
  • Embodiment P16 The antibody of embodiment P15, wherein the inhibition of Wnt5a activity is indicated by the inhibition RhoA and/or Rac1 activity.
  • Embodiment P17 The antibody of embodiment P15, wherein inhibition of Wnt5a activity is indicated by decreased or lack of ARHGEF1, ARHGEF2 and/or ARHGEF6 recruitment.
  • Embodiment P18 The antibody of embodiment P13, wherein the antibody blocks the KNG domain of ROR1 from coupling with ROR2.
  • Embodiment P19 A method of treating CLL comprising administering an agent that disrupts ROR1 and ROR2 coupling to a subject in need thereof.
  • Embodiment 1 A method of identifying an inhibitor of ROR1-ROR2 binding, said method comprising: (i) combining a test agent with a ROR1 protein and a ROR2 protein in a reaction vessel; and (ii) detecting a decrease in binding of said ROR1 protein to said ROR2 protein relative to a standard control, thereby identifying an inhibitor of ROR1-ROR2 binding.
  • Embodiment 2 The method of embodiment 1, wherein said detecting comprises detecting a level of said ROR1 protein bound to said ROR2 protein in the presence of said test agent, wherein a decreased level of said ROR1 protein bound to said ROR2 protein relative to a standard control indicates said test agent is an inhibitor of ROR1-ROR2 binding.
  • Embodiment 3 The method of embodiment 1, wherein said detecting comprises detecting a level of unbound ROR1 protein or unbound ROR2 protein in the presence of said test agent, wherein an increased level of unbound ROR1 protein or an increased level of unbound ROR2 protein relative to a standard control indicates said test agent is an inhibitor of ROR1-ROR2 binding.
  • Embodiment 4 The method of one of embodiments 1-3, wherein said ROR1 protein and said ROR2 protein comprise a detectable moiety.
  • Embodiment 5 The method of embodiment 1, wherein said detecting comprises detecting a level of a guanine exchange factor (GEF) or a guanosine triphosphatase (GTPase) in the presence of said test agent, wherein a decreased level of said GEF relative to a standard control indicates said test agent is an inhibitor of ROR1-ROR2 binding.
  • GEF guanine exchange factor
  • GTPase guanosine triphosphatase
  • Embodiment 6 The method of embodiment 5, wherein said GEF is ARHGEF1, ARHGEF2 or ARHGEF6.
  • Embodiment 7 The method of embodiment 5, wherein said GTPase is RhoA or Rac1.
  • Embodiment 8 The method of one of embodiments 5-7, wherein said GEF or GTPase comprises a detectable moiety.
  • Embodiment 9 The method of one of embodiments 1-8, wherein said combining occurs in the presence of a Wnt5a protein.
  • Embodiment 10 The method of one of embodiments 1-9, wherein said reaction vessel is a column comprising a solid support.
  • Embodiment 11 The method of one of embodiments 1-9, wherein said reaction vessel comprises a cell.
  • Embodiment 12 The method of embodiment 11, wherein said ROR1 protein and said ROR2 protein are expressed on the surface of said cell.
  • Embodiment 13 The method of embodiment 11 or 12, wherein said cell is a cancer cell.
  • Embodiment 14 The method of embodiment 13, wherein said cancer cell is a chronic lymphocytic leukemia (CLL) cell.
  • CLL chronic lymphocytic leukemia
  • Embodiment 15 The method of embodiment 14, wherein said CLL cell is a MEC1 cell.
  • Embodiment 16 The method of one of embodiments 11-14, wherein said cell forms part of an in vitro cell culture.
  • Embodiment 17 The method of one of embodiments 11-16, wherein said cell forms part of an organism.
  • Embodiment 18 The method of embodiment 17, wherein said organism is a mammal.
  • Embodiment 19 The method of embodiment 17, wherein said organism is a mouse.
  • Embodiment 20 The method of one of embodiments 1-19, wherein said test agent is an antibody, a small molecule, a peptide, a protein or a nucleic acid.
  • Embodiment 21 The method of one of embodiments 1-17, wherein said test agent is an antibody.
  • Embodiment 22 The method of embodiment 17 or 18, wherein said antibody is an anti-ROR1 antibody.
  • Embodiment 23 The method of embodiment 17 or 18, wherein said antibody is an anti-ROR2 antibody.
  • Embodiment 24 The method of one of embodiments 17-20, wherein said antibody binds to a ROR1 protein or a ROR2 protein.
  • Embodiment 25 The method of embodiment 21, wherein said antibody binds to a KNG domain.
  • Embodiment 26 The method of one of embodiments 17-22, wherein said antibody is a humanized antibody.
  • Embodiment 27 The method of one of embodiments 17-22, wherein said antibody is a chimeric antibody.
  • Embodiment 28 The method of one of embodiments 17-22, wherein said antibody is a scFv.
  • Embodiment 29 An antibody identified by the method of any one of embodiments 1-22.
  • Embodiment 30 A method of inhibiting a ROR1-ROR2 interaction, said method comprising: (i) contacting a compound identified by the method of any one of embodiments 1-22 with a ROR1-ROR2 complex, wherein said complex comprises a ROR1 protein and a ROR2 protein; and (ii) allowing said compound to inhibit the interaction between said ROR1 protein and said ROR2 protein, thereby inhibiting a ROR1-ROR2 interaction.
  • Embodiment 31 A method of treating cancer in a subject in need thereof, said method comprising administering to a subject a therapeutically effective amount of an antibody, small molecule, peptide or nucleic acid identified by a method of one of embodiments 1-22.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210371535A1 (en) * 2015-04-24 2021-12-02 The Regents Of The University Of California Modulators of ror1-ror2 binding

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10704021B2 (en) 2012-03-15 2020-07-07 Flodesign Sonics, Inc. Acoustic perfusion devices
US10967298B2 (en) 2012-03-15 2021-04-06 Flodesign Sonics, Inc. Driver and control for variable impedence load
US9950282B2 (en) 2012-03-15 2018-04-24 Flodesign Sonics, Inc. Electronic configuration and control for acoustic standing wave generation
US9458450B2 (en) 2012-03-15 2016-10-04 Flodesign Sonics, Inc. Acoustophoretic separation technology using multi-dimensional standing waves
EP3092049A1 (fr) 2014-01-08 2016-11-16 Flodesign Sonics Inc. Dispositif d'acoustophorèse avec double chambre acoustophorétique
US11377651B2 (en) 2016-10-19 2022-07-05 Flodesign Sonics, Inc. Cell therapy processes utilizing acoustophoresis
US11420136B2 (en) 2016-10-19 2022-08-23 Flodesign Sonics, Inc. Affinity cell extraction by acoustics
US11021699B2 (en) 2015-04-29 2021-06-01 FioDesign Sonics, Inc. Separation using angled acoustic waves
US11708572B2 (en) 2015-04-29 2023-07-25 Flodesign Sonics, Inc. Acoustic cell separation techniques and processes
US11474085B2 (en) 2015-07-28 2022-10-18 Flodesign Sonics, Inc. Expanded bed affinity selection
US11459540B2 (en) 2015-07-28 2022-10-04 Flodesign Sonics, Inc. Expanded bed affinity selection
EA201891066A1 (ru) 2015-10-30 2018-10-31 ЭнБиИ-ТЕРАПЬЮТИКС АГ Антитела к ror1
KR20180099887A (ko) 2016-01-20 2018-09-05 더 스크립스 리서치 인스티튜트 Ｒｏｒ2 항체 조성물 및 관련 방법
WO2017127664A1 (fr) 2016-01-20 2017-07-27 The Scripps Research Institute Compositions d'anticorps anti-ror1 et procédés associés
US11214789B2 (en) 2016-05-03 2022-01-04 Flodesign Sonics, Inc. Concentration and washing of particles with acoustics
US11085035B2 (en) 2016-05-03 2021-08-10 Flodesign Sonics, Inc. Therapeutic cell washing, concentration, and separation utilizing acoustophoresis
SG11202000846WA (en) 2017-08-07 2020-02-27 Nbe Therapeutics Ag Anthracycline-based antibody drug conjugates having high in vivo tolerability
BR112020009889A2 (pt) 2017-12-14 2020-11-03 Flodesign Sonics, Inc. acionador e controlador de transdutor acústico
CN111378040B (zh) * 2018-12-29 2021-08-10 深圳大学 检测多种恶性肿瘤细胞的抗体及其应用
CN111378039B (zh) * 2018-12-29 2021-08-24 深圳大学 治疗恶性肿瘤的抗体及其应用
CN113597327A (zh) * 2019-01-29 2021-11-02 加利福尼亚大学董事会 青光眼的新疗法
CN114200142A (zh) * 2021-11-18 2022-03-18 江苏大学 一种检测外泌体Wnt5a蛋白的ELISA试剂盒

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020155121A1 (en) * 1999-10-08 2002-10-24 Devico Anthony Louis Virus coat protein/receptor chimeras and methods of use
US20130289121A1 (en) 2011-01-14 2013-10-31 The Regents Of The University Of Colorado, A Body Corporate Pulmonary disease treatment and diagnosis based on arhgef1
EP2659910A1 (fr) 2010-12-27 2013-11-06 National University Corporation Nagoya University Méthode d'inhibition du signalement pro-survie faisant intervenir les récepteurs tyrosines kinases dans les cellules cancéreuses
WO2014031174A1 (fr) 2012-08-24 2014-02-27 The Regents Of The University Of California Anticorps et vaccins utilisables en vue du traitement de cancers ror1 et de l'inhibition de la métastase

Family Cites Families (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4911920A (en) 1986-07-30 1990-03-27 Alcon Laboratories, Inc. Sustained release, comfort formulation for glaucoma therapy
FR2588189B1 (fr) 1985-10-03 1988-12-02 Merck Sharp & Dohme Composition pharmaceutique de type a transition de phase liquide-gel
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
WO1991010741A1 (fr) 1990-01-12 1991-07-25 Cell Genesys, Inc. Generation d'anticorps xenogeniques
US5427908A (en) 1990-05-01 1995-06-27 Affymax Technologies N.V. Recombinant library screening methods
GB9015198D0 (en) 1990-07-10 1990-08-29 Brien Caroline J O Binding substance
WO1992020791A1 (fr) 1990-07-10 1992-11-26 Cambridge Antibody Technology Limited Methode de production de chainons de paires de liaison specifique
ATE141502T1 (de) 1991-01-15 1996-09-15 Alcon Lab Inc Verwendung von karrageenan in topischen ophthalmologischen zusammensetzungen
US5212162A (en) 1991-03-27 1993-05-18 Alcon Laboratories, Inc. Use of combinations gelling polysaccharides and finely divided drug carrier substrates in topical ophthalmic compositions
CA2124967C (fr) 1991-12-17 2008-04-08 Nils Lonberg Animaux transgeniques non humains capables de produire des anticorps heterologues
US5639641A (en) 1992-09-09 1997-06-17 Immunogen Inc. Resurfacing of rodent antibodies
US6066718A (en) 1992-09-25 2000-05-23 Novartis Corporation Reshaped monoclonal antibodies against an immunoglobulin isotype
US6489137B2 (en) * 1997-06-11 2002-12-03 Chiron Corporation Detection of loss of the wild-type huBUB1 gene
CA2368216A1 (fr) * 1999-03-25 2000-10-05 Banyu Pharmaceutical Co., Ltd. Nouvelles proteines de recepteur couplees aux proteines de liaison a la guanosine triphosphate (gtp), bg3
WO2004006955A1 (fr) 2001-07-12 2004-01-22 Jefferson Foote Anticorps super humanises
US9242014B2 (en) * 2010-06-15 2016-01-26 The Regents Of The University Of California Receptor tyrosine kinase-like orphan receptor 1 (ROR1) single chain Fv antibody fragment conjugates and methods of use thereof
CN103667492B (zh) * 2013-12-13 2015-02-25 青岛大学医学院附属医院 一种检测wnt信号通路的试剂、pcr检测方法及其应用
CA2983293C (fr) * 2015-04-24 2023-06-20 The Regents Of The University Of California Modulateurs de la liaison ror1-ror2

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020155121A1 (en) * 1999-10-08 2002-10-24 Devico Anthony Louis Virus coat protein/receptor chimeras and methods of use
EP2659910A1 (fr) 2010-12-27 2013-11-06 National University Corporation Nagoya University Méthode d'inhibition du signalement pro-survie faisant intervenir les récepteurs tyrosines kinases dans les cellules cancéreuses
US20130289121A1 (en) 2011-01-14 2013-10-31 The Regents Of The University Of Colorado, A Body Corporate Pulmonary disease treatment and diagnosis based on arhgef1
WO2014031174A1 (fr) 2012-08-24 2014-02-27 The Regents Of The University Of California Anticorps et vaccins utilisables en vue du traitement de cancers ror1 et de l'inhibition de la métastase

Non-Patent Citations (26)

* Cited by examiner, † Cited by third party
Title
Anastas, J.N. et al. (Jan. 2013). "WNT signalling pathways as therapeutic targets in cancer," Nat Rev Cancer 13(1):11-26.
Baskar, S. et al. (Jan. 15, 2008). "Unique cell surface expression of receptor tyrosine kinase ROR1 in human B-cell chronic lymphocytic leukemia," Clin Cancer Res 1492):396-404.
Bos, J.L. et al. (Jun. 1, 2007). "GEFs and GAPs: critical elements in the control of small G proteins," Cell 129(5):865-877.
Broome, H.E. et al. (Oct. 2011, e-published Aug. 2, 2011). "ROR1 is expressed on hematogones (non-neoplastic human B-lymphocyte precursors) and a minority of precursor-B acute lymphoblastic leukemia," Leuk Res 35(10):1390-1394.
Cierpicki, T. et al. (Jan. 2015). "Targeting protein-protein interactions in hematologic malignancies: still a challenge or a great opportunity for future therapies?" Immunol Rev 263(1):279-301.
Cook et al., Oncogene, 2014, 33:4021-4035. *
Cui, B. et al. (Jun. 15, 2013). "Targeting ROR1 inhibits epithelial-mesenchymal transition and metastasis," Cancer Res 73(12):3649-3660.
Daneshmanesh, A.H. et al. (Sep. 1, 2008). "Ror1, a cell surface receptor tyrosine kinase is expressed in chronic lymphocytic leukemia and may serve as a putative target for therapy," Int J Cancer 123(5):1190-1195.
Extended European Search Report dated Aug. 8, 2018, for EP Patent Application No. 16784106.3, 11 pages.
Fukuda, T. et al. (Feb. 2008, e-published Feb. 19, 2008). "Antisera induced by infusions of autologous Ad-CD154-leukemia B cells identify ROR1 as an oncofetal antigen and receptor for Wnt5a," PNAS USA 105(8):3047-3052.
Gentile, A. et al. (Apr. 15, 2011, e-published Apr. 12, 2011). "Ror1 is a pseudokinase that is crucial for Met-driven tumorigenesis," Cancer Res 71(8):3132-3141.
Henry, C. et al. (Nov. 24, 2015). "Targeting the ROR1 and ROR2 receptors in epithelial ovarian cancer inhibits cell migration and invasion," Oncotarget 6(37):40310-40326.
International Search Report dated Nov. 15, 2016, for PCT Application No. PCT/US2016/029250, filed Apr. 25, 2016, 5 pages.
Katoh, M. et al. (Nov. 2005). "Comparative genomics on ROR1 and ROR2 orthologs," Onco Rep 14(5):1381-1384.
Niehrs, C. (Dec. 2012, e-published Nov. 15, 2012). "The complex world of WNT receptor signalling," Nat Rev Mol Cell Biol 13(12):767-779.
Oishi, I. et al. (Jan. 1999). "Spatio-temporally regulated expression of receptor tyrosine kinases, mRor1, mRor2, during mouse development: implications in development and function of the nervous system," Genes Cell 4(1):41-56.
Paganoni et al, (Neuroscience 2010, 165(4): 1261, pp. 1-27. *
Paganoni, S. et al. (Feb. 17, 2010, e-published Dec. 1, 2009). "Ror1-Ror2 complexes modulate synapse formation in hippocampal neurons," Neuroscience 165(4):1261-1274.
Rebagay, G. et al. (Apr. 18, 2012). "ROR1 and ROR2 in Human Malignancies: Potentials for Targeted Therapy," Front Oncol 2:34.
Toledo, E.M. et al. (Nov. 2008, e-published Aug. 19, 2008). "Wnt signaling in neuroprotection and stem cell differentiation," Prog Neurobiol 86(3):281-296.
Written Opinion dated Nov. 15, 2016, for PCT Application No. PCT/US2016/029250, filed Apr. 25, 2016, 7 pages.
Yamaguchi, T. et al. (Mar. 20, 2012). "NKX2-1/TITF1/TTF-1-Induced ROR1 is required to sustain EGFR survival signaling in lung adenocarcinoma," Cancer Cell 21(3):348-361.
Yu, J. et al. (Feb. 2016). "Wnt5a induces ROR1/ROR2 heterooligomerization to enhance leukemia chemotaxis and proliferation," J Clin Invest 126(2):585-598.
Zhang et al., The American Journal of Pathology, 2012, 181(6): 1903-1910. *
Zhang, S. et al. (Dec. 2, 2014, e-published Nov. 19, 2014). "Ovarian cancer stem cells express ROR1, which can be targeted for anti-cancer-stem-cell therapy," PNAS USA 111(48):17266-17271.
Zhang, S. et al. (Dec. 2012, e-published Oct. 4, 2012). "The onco-embryonic antigen ROR1 is expressed by a variety of human cancers," Am, J Pathol 181(6):1903-1910.

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210371535A1 (en) * 2015-04-24 2021-12-02 The Regents Of The University Of California Modulators of ror1-ror2 binding

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